Anti-fungals targeting the synthesis of fungal shingolipids

ABSTRACT

The present invention provides a compound having the structure:

This application claims priority of U.S. Provisional Application Nos.62/192,459, filed Jul. 14, 2015 and 62/088,914, filed Dec. 8, 2014, thecontents of each of which are hereby incorporated by reference.

This invention was made with government support under grant numbersAI100631, AI056168, AI071142, and AI087541 awarded by the NationalInstitutes of Health. The government has certain rights in the invention

Throughout this application, certain publications are referenced inparentheses. Full citations for these publications may be foundimmediately preceding the claims. The disclosures of these publicationsin their entireties are hereby incorporated by reference into thisapplication in order to describe more fully the state of the art towhich this invention relates.

BACKGROUND OF THE INVENTION

Globally, over 300 million people are afflicted by a serious fungalinfection and 25 million are at risk of dying or losing their sight(Fungal Infection Trust 2011). Among fungal infections, invasive fungalinfections such as cryptococcosis, candidiasis, aspergillosis andpneumocystosis are the most common and the most life-threatening (Brown,G. D. et al. 2012; Gullo, A. 2009; Tuite, N. L. & Lacey. K. 2013). Theseinfections have risen dramatically over the last 20 years, some over14-fold. The CDC estimates that more than 1 million new cases per yearof cryptococcosis will occur worldwide in patients with AIDS, and600,000 will die from the infection. This is a drastic increaseconsidering that prior to the mid-1950s, fewer than 300 cases had beenreported in the medical literature (reviewed in Sorrell, T. C. et al.2011). Certain medical devices, such as catheters, provide the port ofentry to fungi that colonize the skin and mucosa. As a result,disseminated candidiasis is the 4th most common hospital-acquired sepsiswith >120,000 deaths/year (Perlroth, J. et al. 2007; Rueping, M. J. etal. 2009; Guery B. P. et al. 2009). Disseminated aspergillosisrepresents another invasive fungal infection that is steadily increasingin immunocompromised patients with a mortality rate of 450,000/year(Mayr, A. & Lass-Florl, C. 2011; Maschmeyer, G. et al. 2007; Munoz, P.et al. 2008; Ruping, M. J. et al. 2008). Aspergillus spp. is alsoresponsible for severe asthma by fungal sensitization (SAFS) accountingfor 100,000 additional deaths annually.

Pneumocystis spp. are a group of host-specific opportunistic fungi thatreside in the lungs of humans and animals in nature. The organism isnamed P. jirovecii in humans, P. carinii in rats, and P. murina in mice.Pneumocystis pneumonia (PCP) remains the most prevalent opportunisticinfection in patients infected with the human immunodeficiency virus(HIV). An estimated 539 million patients were discharged from hospitalsbetween 1986 and 2005, of whom an estimated 312,411 had AIDS-associatedPCP. Although numbers of cases of PCP has decreased in economicallydeveloped countries, the worldwide incidence is estimated to exceed400,000 (Kelley, C. F. et al. 2009). Reports on mortality rates for PCPare variable, ranging from 13% to as high as 80%, which even at thelowest rate results in more than 52,000 deaths per year (Kelley, C. F.et al. 2009). PCP is also prevalent in other patient groups, notablypatients that are chronically immune suppressed due to solid organtransplantation or due to chemotherapy for cancer or autoimmune disease.P. jirovecii (Pj) is also a frequent colonizer of the respiratory tractin immunocompetent individuals with other underlying pulmonary diseases,such as Chronic Obstructive Pulmonary Disease (COPD), in which itinitiates a deleterious inflammatory reaction (Huang, L. et al. 2006).Based on these reports, over 1,300,000 people are estimated to die everyyear because of invasive fungal infections and, most likely, this is anunderestimated figure (1, 2). This mortality rate is similar to the onefrom malaria (1,240,000/year) (WHO World Malaria Report 2013) andtuberculosis (1,400,000/year) (WHO World Global Tuberculosis 2013).

While there are about 30 branded prescription antifungal drugs on themarket, three classes of antifungals are mainly used to manage invasivefungal infections: 1) Azoles, such as fluconazole launched in themid-1980s, 2) polyenes, such as amphotericin B launched in the mid-1950sand 3) echinocandins, such as caspofungin launched in early 2000.However, the increased use of current azoles has led to an increase indrug resistance, limiting their effectiveness. In addition, drug-druginteraction issues can be a major impediment to the use of voriconazole,itraconazole and posaconazole. The interactions with cancer chemotherapyagents and immunosuppressants can be particularly difficult to handleclinically. Systemic antifungals, such as amphotericin B, tend to haverelatively high toxicity and side effects. The echinocandins have alower incidence of adverse events compared to older antifungals but theybind highly to serum proteins, there are no oral formulations, and theirantifungal spectrum of activity is very narrow (Farowski, F. et al.2012; Farowski, F. et al. 2013; Odabasi, Z. et al. 2007; Saribas, Z. etal. 2012; Yanni, S. B. et al. 2011). In the case of Pneumocystis, thesituation is direr. Pneumocystis pneumonia does not respond to any ofthe standard antifungals described above (Carmona, E. M. & Limper, A. H.2011). The drug of choice for the treatment and chemoprophylaxis of PCPis trimethoprim-sulfamethoxazoe (TMP-SMX). Analysis of P. jiroveciiisolates demonstrates that the pathogen is evolving mutations in thetarget genes of TMP-SMX, suggesting P. jirovecii could soon becomeresistant to SMX in the combination, considered the more potent of thetwo drugs that makeup the combination therapy (Ma, L. et al. 1999).Atovaquone and pentamidine, both second line treatments, suffer from lowefficacy and severe adverse events (SAEs) that include nephrotoxicity,neutropenia, hypotension and hypoglycemia (Benfield, T. et al. 2008).Atovaquone inhibits the mitochondrial cytochrome Bc1 complex inparasites at much lower concentrations than the respective mammaliancomplex. However, evolving resistance to atovaquone, corresponding tomutations in the Pneumocystis cytochrome b gene, has been observed(Kazanjian, P. et al. 2001). Pentamidine has a broad antimicrobialaction with no specific target known and is highly toxic and oftenconsidered to be a drug of last resort. We are faced, then, with agrowing patient population, a microorganism that cannot be easilysubjected to detailed biochemical analysis in the laboratory, adeveloping resistance to standard of care medications and a limitedindustrial effort to advance new therapies into the clinic. Thus, thereis a need for new, safer and more effective compounds.

Studies in our and other laboratories identified sphingolipids as keyregulators of fungal pathogenesis (reviewed in Heung, L. J. et al. 2006and Singh, A. 2011). Particularly, a fungal sphingolipid namedglucosylceramide (GlcCer) is required for the pathogenic fungusCryptococcus neoformans to cause a lethal meningo-encephalitis(Kechichian, T. B. et al. 2007; Rittershaus, P. C. et al. 2006). Infact, mice survived the infection by a C. neoformans mutant strainlacking the final enzyme for the synthesis of GlcCer (GlcCer synthase 1or Gcs1). The Δgcs1 mutant was confined in the lung granuloma and it didnot reach the bloodstream and, thus, it did not disseminate to thebrain. Later, other investigators corroborated and extended our findingsthat mutation of genes involved in the last steps of the GlcCer pathwayaffect fungal virulence not only of fungi infecting humans, such as C.neoformans (Liu, O. W. et al. 2008; Singh, A. et al. 2012), Candidaalbicans (Oura, T. & Kajiwara, S. 2010; Noble, S. M. et al. 2010; Oura,T. & Kajiwara, S. 2008), and Aspergillus fumigatus (Levery, S. B. et al.2002), but also of fungi infecting plants (da Silva, A. F. et al. 2004;Ramaoorthy, V. et al. 2009). That GlcCer is required for fungalvirulence in plants is also suggested by studies showing that plantsdefend themselves against fungi by producing specific defensins (e.g.RsAFP2 and others) that bind to fungal and not mammalian GlcCer(Thevissen, K. et al. 2004). Interestingly, these plant defensins areable to bind GlcCer of human pathogenic fungi and able to kill them invitro and, in some cases, during in vivo infection in animal models(Thevissen, K. 2004; Tavares, P M. 2008; Aerts, A M. 2007; Lobo, D S.2007; Thevissen, K. 2012; Mello Ede, O. et al. 2014; Oguro, Y. et al.2014; Goncalves, S. et al. 2012; de Medeiros, L. N. et al. 2010).

Mechanistic studies revealed that GlcCer is involved in the regulationof fungal cell replication in environments characterized byneutral/alkaline pH (Kechichian, T. B. et al. 2007; Levery, S. B. et al.2002; Rhome, R. et al. 2011). Particularly, when fungal cells lackingGlcCer are exposed to neutral/alkaline pH, they cannot progress throughthe cell cycle and, thus, cytokinesis does not occur (Rittershaus, P. C.et al. 2006; Levery, S. B. et al. 2002; Saito, K. et al. 2006). Later,we linked this phenomenon to the regulation by GlcCer of physicalproperties of fungal plasma membranes of C. neoformans (Singh, A. et al.2012). The synthesis of GlcCer seems to be important also duringPneumocystis pneumonia (PCP) as GlcCer synthase transcripts have beenfound to be elevated at the time of isolation of the fungus from afulminate lung infection (Cushion, M. T. et al. 2007). Interestingly, inmost dimorphic fungi, production of GlcCer is detected only in the hostinfective form (yeast) and not in the environmental form (mold)(Warnecke, D. et al. 2003; Rhome, R. et al. 2007; Toledo, M. S. et al.2001). Taken together, these studies suggest that GlcCer is most likelya pan-fungal virulence factor required during infection to promotefungal growth at neutral/alkaline environments in the host (e.g.alveolar spaces, cerebrospinal fluid and bloodstream), and as such, itis a promising novel drug target. Currently, inhibitors that block thefungal but not the mammalian GlcCer synthesis are not available.

SUMMARY OF THE INVENTION

The present invention provides a compound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, and R₆, and R₇ are each independently —H, halogen,        CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂,        —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₉, R₁₀, and R₁₁ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅; and    -   R₈ and R₁₂ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OAc, —COR₁₃,        —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, NR₁₄R₁₅,        —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   wherein when R₁, R₂, R₃, R₄, R₅, R₆, R₈, R₁₁, R₁₂ are each —H,        R₉ is —Br, and R₁₀ is —OH, then R₇ is other than —CH₃,    -   or a pharmaceutically acceptable salt or ester thereof.

The present invention also provides a method of inhibiting the growth ofa fungus comprising contacting the fungus with an effective amount of acompound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,        —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NHCOR₁₂, or —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   when R₁, R₂, R₃, R₄, R₅, R₆, R₈, R₁₁, R₁₂ are each —H, R₉ is        —Br, and —R₁₀ is —OH or —OCH₃, then R₇ is other than —CH₃; and    -   when R₁, R₂, R₃, R₄, R₅, R₈, R₉, R₁₁, and R₁₂ are each —H, and        —R₁₀ is OH, then R₆ and R₇ are other than —H and —CH₃ or —Br and        H, respectively,    -   or a pharmaceutically acceptable salt or ester thereof, so as to        thereby inhibit the growth of the fungus.

The present invention also provides a method of inhibiting fungalshingolipid synthesis in a fungus comprising contacting the fungus withan effective amount of a compound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,        —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NHCOR₁₂, or —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   when R₁, R₂, R₃, R₄, R₅, R₆, R₈, R₁₁, R₁₂ are each —H, R₉ is        —Br, and —R₁₀ is —OH or —OCH₃, then R₇ is other than —CH₃; and        when R₁, R₂, R₃, R₄, R₅, R₈, R₉, R₁₁, and R₁₂ are each —H, and        —R₁₀ is OH, then R₆ and R₇ are other than —H and —CH₃ or —Br and        H, respectively,    -   or a pharmaceutically acceptable salt or ester thereof, so as to        thereby inhibit shingolipid synthesis in the fungus.

The present invention also provides a method of inhibiting fungalshingolipid synthesis in a fungus in a mammal without substantiallyinhibiting mammalian shingolipid synthesis comprising administering tothe mammal an effective amount of a compound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,        —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NHCOR₁₂, or —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   when R₁, R₂, R₃, R₄, R₅, R₆, R₈, R₁₁, R₁₂ are each —H, R₉ is        —Br, and —R₁₀ is —OH or —OCH₃, then R₇ is other than —CH₃; and        when R₁, R₂, R₃, R₄, R₅, R₈, R₉, R₁₁, and R₁₂ are each —H, and        —R₁₀ is OH, then R₆ and R₇ are other than —H and —CH₃ or —Br and        H, respectively,    -   or a pharmaceutically acceptable salt or ester thereof, so as to        thereby inhibit fungal shingolipid synthesis in the fungus in        the mammal without substantially inhibiting mammalian        shingolipid synthesis.

The present invention further provides method of inhibiting the growthof a fungus comprising contacting the fungus with an effective amount ofa compound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,        —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or        —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt or ester thereof, so as to        thereby inhibit the growth of the fungus,    -   wherein the fungus is other than Cryptococcus neoformans.

The present invention further provides a method of inhibiting fungalshingolipid synthesis in a fungus comprising contacting the fungus withan effective amount of a compound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,        —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or        —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt or ester thereof, so as to        thereby inhibit shingolipid synthesis in the fungus,    -   wherein the fungus is other than Cryptococcus neoformans.

The present invention further provides a method of inhibiting fungalshingolipid synthesis in a fungus in a mammal without substantiallyinhibiting mammalian shingolipid synthesis comprising administering tothe mammal an effective amount of a compound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,        —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or        —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt or ester thereof, so as to        thereby inhibit fungal shingolipid synthesis in the fungus in        the mammal without substantially inhibiting mammalian        shingolipid synthesis,    -   wherein the fungus is other than Cryptococcus neoformans.

The present invention furthermore provides a method of inhibiting thegrowth of or killing a fungus in a subject afflicted with a fungalinfection comprising administering to the subject an effective amount ofa compound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,        —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NHCOR₁₂, or —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   when R₁, R₂, R₃, R₄, R₅, R₆, R₈, R₁₁, R₁₂ are each —H, R₉ is        —Br, and —R₁₀ is —OCH₃, then R₇ is other than —CH₃; and when R₁,        R₂, R₃, R₄, R₅, R₈, R₉, R₁₁, and R₁₂ are each —H, and —R₁₀ is        OH, then R₆ and R₇ are other than —H and —CH₃ or —Br and H,        respectively,    -   or a pharmaceutically acceptable salt or ester thereof, so as to        thereby inhibiting the growth of or kill the fungus in the        subject afflicted with the fungal infection.

The present invention also provides a method of inhibiting the growth ofor killing a fungus in a subject afflicted with a fungal infectioncomprising administering to the subject an effective amount of acompound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,        —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or        —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt or ester thereof, so as to        thereby inhibit the growth of or kill the fungus in the subject        afflicted with the fungal infection,    -   wherein the fungus in the subject afflicted with the fungal        infection is other than Cryptococcus neoformans.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A: BHBM and 1 inhibit the synthesis of fungal but not mammalianglucosylceramide. Thin layer chromatography analysis of the synthesis ofglucosylceramide (GlcCer) upon in vivo labeling of C. neoformans (Cn) orJ774 cells with ³H-palmitate and treated with BHBM or 1 at the indicatedconcentrations.

FIG. 1B: Structure ofN′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and3-bromo-N′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (1). MIC.Minimum inhibitory concentration; MFC, minimum fungicidal concentration.

FIG. 2A: Killing activity of BHBM and 1. Killing activity was determinedusing a killing in vitro assay in which the compounds, at theillustrated concentrations, were added to C. neoformans cells and tubesincubated at 37° C., 5% CO₂ and pH 7.4. Colony forming units (CFU) werecounted during the course of 96 hours of incubation.

FIG. 2B: 1 showed higher killing activity than BHBM (A). *, P<0.05, 1μg/ml of BHBM at 48 h versus 0.25 μg/ml, 1 or 4 μg/ml at 72 h versus0.25 μg/ml, 4 μg/ml at 96 h versus 0.25 μg/ml; # P<0.05, 1 or 4 μg/ml of1 at 24 h versus 0.25 μg/ml or drug. Cn, C. neoformans.

FIG. 2C: Intracellular activity of BHBM was assessed by incubatingmacrophages internalized with C. neoformans with different concentrationof BHBM in absence of opsonins. *, P<0.05, extracellular orintracellular treated (0.25, 1 or 4 μg/ml) versus extracellular orintracellular untreated (0 μg/ml), respectively. Statistical analysiswas performed using the analysis of variance (ANOVA). Statisticalsignificance is accepted at a P value <0.05.

FIG. 3A: Effect of BHBM and 1 on cryptococcosis. Survival of miceinfected with C. neoformans (Cn) and receiving 1.2 mg/Kg/day of eitherBHBM or 1 intraperitoneally. *P<0.01, BHBM or 1 treated brains versusuntreated. Statistical analysis for survival studies was performed usingStudent-Newman-Keuls t test for multiple comparisons using INSTAT.

FIG. 3B: Tissue burden culture of lungs and brains of infected mice wereperformed at day 25 (untreated) or at day 60 (treated). *P<0.001, BHBMor 1 treated brains versus untreated brains; # P<0.01, BHBM or 1 treatedlungs versus untreated lungs. Statistical analysis for CFU data wasperformed using the analysis of variance (ANOVA).

FIG. 3C-F: Histopathology by H&E staining of lungs and brains ofuntreated mice (w/o BHBM or 1) at day 25 and treated mice (+BHBM or +1)at day 60 of infection. Lungs and brain tissue of untreated mice showedextensive cryptococcal infiltration (arrows in (a) and (b),respectively). Lungs of mice treated with BHBM (c) or 1 (e) show foci ofCn cells limited to few regions of the lung whereas no fungal cells wereobserved in brains treated with either BHBM (d) or 1 (f). Scale bar: 150μm. Panels (a), (c) and (e)=lung. Panels (b), (d) and (f)=brain.

FIG. 4A: Effect of BHBM and 1 on pneumocystosis. Survival ofcorticosteroid-immunosuppressed mice after 13 days of BHBM treatment. Asignificant difference was observed between the BHBM high dose and BHBMlow dose treatment groups. C/S, control steroids vehicle treated mice,T/S, Trimethoprim/Sulfamethaxozole.

FIG. 4B-C: Mean asci (B) and nuclei (C) counts after 13 days of BHBMtreatment. (C/S) vehicle, negative control. T/S,trimethoprim/Sulfamethaxozole.

FIG. 4D: Survival curves after 14 days of 1 treatment. C/S, controlsteroids vehicle immunosuppressed mice.

FIG. 4E-F: Mean asci (E) and nuclei (F) counts after 14 days of 1treatment. *P<0.01, Statistical analysis for survival studies wasperformed using Student-Newman-Keuls t test for multiple comparisonsusing INSTAT. Statistical analysis for trophic form and asci counts wasperformed using the analysis of variance (ANOVA).

FIG. 5A: Effect of BHBM and 1 on candidiasis. Survival of mice infectedwith C. albicans SC 5314 and receiving 1.2 mg/Kg/day of either BHBM or 1intraperitoneally. *P<0.01, BHBM or 1 treated brains versus untreated.Statistical analysis for survival studies was performed usingStudent-Newman-Keuls t test for multiple comparisons using INSTAT.

FIG. 5B: Tissue burden culture of organs of infected mice were performedat day 21 of treated mice that survived the infection.

FIG. 6A: Measurements of C. neoformans sphingolipids upon treatment withBHBM. Thin layer chromatography of sphingolipids of untreated orBHBM-treated C. neoformans cells after in vivo labeling with ³Hpalmitate.

FIG. 6B-D: Mass spectrometry analysis of sphingosines (sphingosine anddihydrosphingosine—SPHs (B), C18 dihydroceramide—C18dhCER (C), and 9methyl glucosylceramide (GlcCer) (D) in treated and untreated Cn cells.*, P<0.05 versus untreated (no drug) cultures. Statistical analysis forCFU data was performed using the analysis of variance (ANOVA).Statistical significance is accepted at a P value <0.05.

FIG. 7A: Effects of BHBM on Golgi morphology and vesicle secretion in C.neoformans and C. albicans. A) Control (CT) or BHBM-treated cells werestained with C6-NBD-ceramide (Golgi, green fluorescence or gray in black& white depection) and DAPI (nucleus, blue fluorescence or light gray inblack & white depection). Columns 1 and 2 show C. neoformans, whilecolumns 3 and 4 show C. albicans cells.

FIG. 7B-C: Quantitative determination of vesicular sterols in C.neoformans (B) and C. albicans (C) secreted vesicles revealed that BHBMtreatment efficiently inhibited vesicle secretion. *P<0.05. WF, whitefield; NBD-Cer, NBD-ceramide.

FIG. 8: Schematic of high-throughput screening using the DiverSet-CLChemBridge Library. Compounds were screened using microtiter plates (96wells) in which 2×10⁴ Cryptococcus neoformans (Cn) H99 strains cellswere added to 150 μM individual compound concentration per well in YNBmedium with 1.5% DMSO. Compounds were selected for activity if inhibitedgrowth of Cn H99≥80%. Compounds were considered inactive if there was nodifference between drug-positive well and drug negative well.Drug-negative well contained 2×10⁴ Cn H99 in YNB medium with 1.5% DMSO.

FIG. 9: Structure, solubility and stability of BHBM.

FIG. 10: Structure, solubility and stability of 1.

FIG. 11: Synergistic activity of BHBM and 1 with fluconazole,amphotericin B, caspofungin, tunicamycin and aureobasidin A.

FIG. 12: BHBM has no effect on bacterial cell. E. Coli or P. aeruginosa(6×10⁷ cells) were spread onto a LB agar plate. Several wells werepunched out using a 1 mL cut-pipet tip and either BHBM, ampicillin orgentamicin at the illustrated concentration was added to the well andthe plate was incubated at 30° C. for 24 hr. No zone of inhibition couldbe seen up to 160 μg/mL BHBM on E. coli as well as P. aeruginosa.Ampicillin and Gentamicin were used as positive controls.

FIG. 13: BHBM and 1 do not induce drug resistance in vivo. Cn cells werepassaged daily in either BHBM or 1 at the illustrated concentrations(below MIC₈₀) for 15 days. At 0, 5, 10, and 15 days, cells were testedfor MIC₈₀. Fluconazole was used as positive control.

FIG. 14: Liver and kidney function test in the blood of BHBM treatedmice that received 1.2 mg/kg/day of BHBM intraperitoneally for 60 days.

FIG. 15: Total leukocytes counts in the blood of BHBM treated andun-infected mice. The mice received 1.2 mg/kg/day of BHBMintraperitoneally for 60 days.

FIG. 16: Erythrocytes and leukocytes measurements in the blood of BHBMtreated and un-infected mice. The mice received 1.2 mg/kg/day of BHBMintraperitoneally for 60 days.

FIG. 17: In vitro toxicity studies at 24 and 48 hr after exposure toBHBM or 1 using dimethylthiazol-diphenyl tetrazolium bromide (MTT) assayin J774 mammalian cells.

FIG. 18: In vitro toxicity studies at 24 hr of dexamethasone (DEX) aloneor in combination with BHBM or 1 using MTT assay in J774 mammaliancells.

FIG. 19: In vitro toxicity studies at 24 hr of cyclophosphamide (CP)alone or in combination with BHBM or 1 using MTT assay in J774 mammaliancells.

FIG. 20A: Survival of CD-4-depleted immunosuppressed mice after 14 daysof treatment with 1. Treatment showed improvement in survival. Noevidence of drug toxicity was observed. C/S, control vehicleCD4-depleted mice.

FIG. 20B-C: Mean asci (b) and nuclei (c) count after 14 days oftreatment. *P<0.05. Statistical analysis for survival studies wasperformed using Student-Newman-Keuls t test for multiple comparisonsusing INSTAT. Statistical analysis for trophic form and asci counts wasperformed using analysis of variance (ANOVA).

FIG. 21: Pharmacokinetics of BHBM in mice, Mean blood concentration-timeprofiles of BHBM after a single intravenous (IV) 1.6 mg/kg (high dose)or intraperitoneal (IP) 0.8 mg/kg (low IP dose) or 1.6 mg/kg (high IPdose) administration in normal or immunosuppressed (IS) and infected(IN) mice. N=3 mice per group per time point.

FIG. 22: MIC₈₀ of compounds BHBM and 1-10.

FIG. 23: In vitro activity (MIC₈₀), solubility, stability and toxicityof compounds BHBM and 1-10.

FIG. 24A: Pathway leading to the synthesis of glucosylceramide (GlcCer)in fungal and mammalian cells. GCS, mammalian glucosylceramide synthase;Sld8, shingolipid delta-8 desaturase; Smt1, shingolipid C9 methyltransferase; Gcs1, fungal glucosylceramide synthase.

FIG. 24B: In vitro Gcs1 activity using ¹⁴C-UDP glucose and C16 ceramidein presence of cell lysates extracted from either C. Neoformans (Cn)wild-type (WT) or Cn ΔGcs1 mutant strain in which Gcs1 is deletedwithout or with different concentrations of BHBM.

FIG. 25: Hetero insufficiency profiling and homozygoze insufficiencyprofiling (HIP-HOP) analysis of BHBM. Scatter plot of Saccharomycescerevisiae hterozygote library treated with BHBM. Plot shows few genesclearly involved in sensitivity to BHBM (see Table 3).

FIG. 26: Tritiated palmitate in vivo labeling of J774 cells in thepresence or absence of BHBM or 1. Thin layer chromatography showed nosignificant changes in the synthesis of mammalian GlcCer upon treatment.

FIG. 27: Bone marrow derived macrophages treated with BHBM for threedays. Presence of mammalian GlcCer was detected using FITC labeledcholera toxin subunit B, which binds specifically to mammalian GlcCer.BHBM treatment did not significantly affect GlcCer level.

FIG. 28A-D: Gas ion chromatogram of sterols extracted from untreated andBHBM treated C. Neoformans cells. A, B, C, and D represent the sterolprofiles of 0, 1, 2, and 4 μg/mL BHBM C. Neoformans treated H99wild-type cells, respectively. Lipid extraction was performed asdescribed by Singh et al. (Cell Microbiol. (2012) 14, 4, 500-516).Sterol derivatization, detection, and analysis was performed by GC-MSusing methods described by Nes et al. Arch Biochem. Biophys. (2009) 481,2, 210-218; Singh et al. OMICS (2013) 17, 2, 84-93; and Chang et al.(2014) PLoS Genet. (2014) 10, 4, e1004292. The experiments wereperformed in duplicate. Structures are: 1. squalene; 2. cholesterol(internal standard); 3. dehyoergosterol; 4. ergosterol; 5.ergosta-7,22-dien-3-ol; 6. ergosta-8-ol; 7. Fecosterol; 8. Ergosta-7-il;9. Lanosterol; 10. 4α-methyl episterol; 11.24-methylenelanost-8-en-3-ol; 12. Putative noreburicol; 13., 14., 15.Unknown sterol intermediates.

FIG. 29: Comparison of BHBM and 1 with fluconazole and amphotericin B oncryptococcosis. Survival of mice infected intravenously with C.neoformans. The average survival of the control group (solvent) was7.0±1.1 days. The BHBM, 1 and fluconazole (FLC) groups showed an averagesurvival of 11.1±3.7 (P<0.005), 10.5±4.2 (P<0.01) and 10.0±2.0(P<0.005), respectively. Amphotericin B (AMB) group had an averagesurvival of 23.5±6.5 (P<0.005). Statistical analysis for survivalstudies was performed using Kruskal-Wallis test.

FIG. 30A-D: TEM images of C. neoformans (A) untreated or treated with 4μg/mL of (B) 1 and (C) BHBM for 6 hours. A higher magnification image ofBHBM treated cell is shown in (D). White arrows in G show membranestructure, whereas black arrows indicate intracellular vesicles. Blackscale bar=500 nm in A, B, and C; 200 nm in D. Representative data of 3separate experiments.

FIG. 31A-D: TEM images of C. neoformans treated with 4 μg/mL of BHBM (Aor D) or 1 (B and C) for 6 hours. Giant multivesicular bodies (longwhite arrows are present in A, B, and D containing multiple vesicles(black arrows) which eventually replace the entire cell (D). (C) is ahigher magnification image of a multivascular body in (B). Short arrowsin B illustrate the many vacuoles present in this bud cell. Black scalebar=500 nm in A and B; 100 nm in C and D.

FIG. 32A-C: Effect of BHBM, fluconazole and methyl methane sulfonate(MMS) on wild-type BY4741 and Δap15, Δcos111, Δmkk1 and Δste2 deletionstrains. Relative growth inhibition was calculated by the average rateafter normalizing the OD600 values in drug wells against the DMSOcontrol wells on each assay plate. The mutant strains show increasedresistance to BHBM but not to fluconazole or MMS. Results from twoindependent growth assays.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, and R₆, and R₇ are each independently —H, halogen,        CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂,        —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₉, R₁₀, and R₁₁ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅; and    -   R₈ and R₁₂ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OAc, —COR₁₃,        —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, NR₁₄R₁₅,        —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   wherein when R₁, R₂, R₃, R₄, R₅, R₆, R₈, R₁₁, R₁₂ are each —H,        R₉ is —Br, and R₁₀ is —OH, then R₇ is other than —CH₃,    -   or a pharmaceutically acceptable salt or ester thereof.

In some embodiments, at least one of R₈, R₉, R₁₀, R₁₁, and R₁₂ are otherthan —H. In some embodiments, at least two of R₈, R₉, R₁₀, R₁₁, and R₁₂are other than —H.

In some embodiments, R₇ is C₂-C₁₂ alkyl or C₂-C₂₀ alkyl. In someembodiments, R₁ is H or —CH₃; and R₂ is H or —CH₃. In some embodiments,R₁ is —H; and R₂ is —H.

In some embodiments, the compound wherein

-   -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br, alkyl,        —OH, —OR₁₃, or —NR₁₄R₁₅; and    -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H, —Br, alkyl,        —OH, —OR₁₃, or —NR₁₄R₁₅,        -   wherein each occurrence of R₁₃, R₁₄, and R₁₅ is alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound wherein

-   -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br, or alkyl;        and    -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H, —Br, —OH,        —OR₁₃, or —NR₁₄R₁₅,        -   wherein each occurrence of R₁₃, R₁₄, and R₁₅ is alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound wherein

-   -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br, or —CH₃;        and    -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H, —Br, —OH,        —OCH₃, or —N(CH₃)₂,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound having the structure:

-   -   wherein    -   R₉, R₁₀, and R₁₁ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅; and    -   R₈ and R₁₂ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OAc, —COR₁₃,        —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, NR₁₄R₁₅,        —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound wherein

-   -   R₈ and R₁₂ are each independently —H, —Br, or —NR₁₄R₁₅,    -   R₉, R₁₀, and R₁₁ are each independently —H, —Br, —OH, —OR₁₃, or        —NR₁₄R₁₅,    -   wherein each occurrence of R₁₃, R₁₄, and R₁₅ is alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound wherein

-   -   R₈ and R₁₂ are each independently —H, —Br, or —N(CH₃)₂,    -   R₉, R₁₀, and R₁₁ are each independently —H, —Br, —OH, —OCH₃, or        —N(CH₃)₂,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound having the structure:

-   -   wherein    -   R₃, R₄, R₅, and R₆, and R₇ are each independently —H, halogen,        CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂,        —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound wherein

-   -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br, or alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound wherein

-   -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br, or —CH₃,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound having the structure:

-   -   or a pharmaceutically acceptable salt thereof.

In some embodiments, a pharmaceutical composition comprising thecompound of the present invention and a pharmaceutically acceptablecarrier.

The present invention also provides a method of inhibiting the growth ofa fungus comprising contacting the fungus with an effective amount of acompound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,        —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NHCOR₁₂, or —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   when R₁, R₂, R₃, R₄, R₅, R₆, R₈, R₁₁, R₁₂ are each —H, R₉ is        —Br, and —R₁₀ is —OH or —OCH₃, then R₇ is other than —CH₃; and    -   when R₁, R₂, R₃, R₄, R₅, R₈, R₉, R₁₁, and R₁₂ are each —H, and        —R₁₀ is OH, then R₆ and R₇ are other than —H and —CH₃ or —Br and        H, respectively,    -   or a pharmaceutically acceptable salt or ester thereof, so as to        thereby inhibit the growth of the fungus.

The present invention also provides a method of inhibiting fungalshingolipid synthesis in a fungus comprising contacting the fungus withan effective amount of a compound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,        —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NHCOR₁₂, or —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   when R₁, R₂, R₃, R₄, R₅, R₆, R₈, R₁₁, R₁₂ are each —H, R₉ is        —Br, and —R₁₀ is —OH or —OCH₃, then R₇ is other than —CH₃; and    -   when R₁, R₂, R₃, R₄, R₅, R₈, R₉, R₁₁, and R₁₂ are each —H, and        —R₁₀ is OH, then R₆ and R₇ are other than —H and —CH₃ or —Br and        H, respectively,    -   or a pharmaceutically acceptable salt or ester thereof, so as to        thereby inhibit shingolipid synthesis in the fungus.

The present invention also provides a method of inhibiting fungalshingolipid synthesis in a fungus in a mammal without substantiallyinhibiting mammalian shingolipid synthesis comprising administering tothe mammal an effective amount of a compound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,        —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NHCOR₁₂, or —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   when R₁, R₂, R₃, R₄, R₅, R₆, R₈, R₁₁, R₁₂ are each —H, R₉ is        —Br, and —R₁₀ is —OH or —OCH₃, then R₇ is other than —CH₃; and    -   when R₁, R₂, R₃, R₄, R₅, R₈, R₉, R₁₁, and R₁₂ are each —H, and        —R₁₀ is OH, then R₆ and R₇ are other than —H and —CH₃ or —Br and        H, respectively,    -   or a pharmaceutically acceptable salt or ester thereof, so as to        thereby inhibit fungal shingolipid synthesis in the fungus in        the mammal without substantially inhibiting mammalian        shingolipid synthesis.

In some embodiments, the method wherein at least one of R₈, R₉, R₁₀,R₁₁, and R₁₂ are other than —H. In some embodiments, the method whereinat least two of R₈, R₉, R₁₀, R₁₁, and R₁₂ are other than —H.

In some embodiments, the method wherein R₁ is H or —CH₃; and R₂ is H or—CH₃. In some embodiments, the method wherein R₁ is —H; and R₂ is —H. Insome embodiments, the method wherein R₇ is C₂-C₁₂ alkyl or C₂-C₂₀ alkyl.

In some embodiments, the method wherein

-   -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br, alkyl,        —OH, —OR₁₃, or —NR₁₄R₁₅; and    -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H, —Br, alkyl,        —OH, or —OR₁₃,        -   wherein each occurrence of R₁₃, R₁₄, and R₁₅ is alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein

-   -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br, or alkyl;        and    -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H, —Br, —OH,        or —OR₁₃,        -   wherein R₁₃ is alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein

-   -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br, or —CH₃;        and    -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H, —Br, —OH,        or —OCH₃,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein the compound has the structure:

-   -   wherein    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NHCOR₁₂, or —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein

-   -   R₈ and R₁₂ are each independently —H, —Br, or —NR₁₄R₁₅,    -   R₉, R₁₀, and R₁₁ are each independently —H, —Br, —OH, or —OR₁₃,        -   wherein each occurrence of R₁₃, R₁₄, and R₁₅ is alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein

-   -   R₈ and R₁₂ are each independently —H, —Br, or —N(CH₃)₂,    -   R₉, R₁₀, and R₁₁ are each independently —H, —Br, —OH, or —OCH₃,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein the compound has the structure:

-   -   wherein    -   R₃, R₄, R₅, and R₆, and R₇ are each independently —H, halogen,        CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂,        —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein

-   -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br, or alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein

-   -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br, or —CH₃,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein the compound has the structure:

-   -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method further comprising contacting the funguswith an amount of an anti-fungal agent.

In some embodiments, the method wherein the amount of the compound andthe amount of the anti-fungal agent when taken together is moreeffective to inhibit the growth of the fungus than the anti-fungal agentalone.

In some embodiments, the method wherein the anti-fungal agent isfluconazole, amphotericin B, caspofungin, tunicamycin or aureobasidin A.

In some embodiments, the method wherein the fungus is CryptococcusNeoformans, Cryptococcus gattii, Candida albicans, Candida krusei,Candida glabrata, Candida parapsilosis, Candida guilliermondii,Aspergillus fumigatus, Rhizopus oryzae, Rhizopus spp., Blastomycesdermatitis, Histoplasma capsulatum, Coccidioides spp., Paecilomycesvariotii, Pneumocystis murina, Pneumocystis jiroveci, Histoplasmacapsulatum, Aspergillus spp., a dimorphic fungi or a mucorales fungi.

In some embodiments, the method wherein the fungal shingolipid isglucosylceramide (GlcCer).

The present invention further provides method of inhibiting the growthof a fungus comprising contacting the fungus with an effective amount ofa compound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,        —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or        —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt or ester thereof, so as to        thereby inhibit the growth of the fungus,    -   wherein the fungus is other than Cryptococcus neoformans.

The present invention further provides a method of inhibiting fungalshingolipid synthesis in a fungus comprising contacting the fungus withan effective amount of a compound having the structure:

wherein

-   -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,        —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or        —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt or ester thereof, so as to        thereby inhibit shingolipid synthesis in the fungus,    -   wherein the fungus is other than Cryptococcus neoformans.

The present invention further provides a method of inhibiting fungalshingolipid synthesis in a fungus in a mammal without substantiallyinhibiting mammalian shingolipid synthesis comprising administering tothe mammal an effective amount of a compound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,        —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or        —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt or ester thereof, so as to        thereby inhibit fungal shingolipid synthesis in the fungus in        the mammal without substantially inhibiting mammalian        shingolipid synthesis,    -   wherein the fungus is other than Cryptococcus neoformans.

In some embodiments, the method wherein at least one of R₈, R₉, R₁₀,R₁₁, and R₁₂ are other than —H. In some embodiments, the method whereinat least two of R₈, R₉, R₁₀, R₁₁, and R₁₂ are other than —H.

In some embodiments, the method wherein R₁ is H or —CH₃; and R₂ is —H or—CH₃. In some embodiments, the method wherein R₁ is —H; and R₂ is —H. Insome embodiments, the method wherein R₇ is C₂-C₁₂ alkyl or C₂-C₂₀ alkyl.

In some embodiments, the method wherein

-   -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br, alkyl,        —OH, —OR₁₃, or —NR₁₄R₁₅; and    -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H, —Br, alkyl,        —OH, —OR₁₃ or NR₁₄R₁₅,        -   wherein each occurrence of R₁₃, R₁₄, and R₁₅ is alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein

-   -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br, or —CH₃;        and    -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H, —Br, —OH,        —OCH₃, or —N(CH₃)₂,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein the compound has the structure:

-   -   wherein    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or        —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein

-   -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H, —Br, alkyl,        —OH, —OR₁₃ or NR₁₄R₁₅,        -   wherein each occurrence of R₁₃, R₁₄, and R₁₅ is alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein

-   -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H, —Br, —OH,        —OCH₃, or —N(CH₃)₂,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein the compound has the structure:

-   -   wherein    -   R₃, R₄, R₅, and R₆, and R₇ are each independently —H, halogen,        CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂,        —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein

-   -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br, or alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein

-   -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br, or —CH₃,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein the compound has the structure:

-   -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method further comprising contacting the funguswith an amount of an anti-fungal agent.

In some embodiments, the method wherein the amount of the compound andthe amount of the anti-fungal agent when taken together is moreeffective to inhibit the growth of the fungus than the anti-fungal agentalone.

In some embodiments, the method wherein the anti-fungal agent isfluconazole, amphotericin B, caspofungin, tunicamycin or aureobasidin A.

In some embodiments, the method wherein the fungus is Cryptococcusgattii, Candida albicans, Candida krusei, Candida glabrata, Candidaparapsilosis, Candida guilliermondii, Aspergillus fumigatus, Rhizopusoryzae, Rhizopus spp., Blastomyces dermatitis, Histoplasma capsulatum,Coccidioides spp., Paecilomyces variotii, Pneumocystis murina,Pneumocystis jiroveci, Histoplasma capsulatum, Aspergillus spp., adimorphic fungi or a mucorales fungi.

In some embodiments, the method wherein the fungal shingolipid isglucosylceramide (GlcCer).

The present invention furthermore provides a method of inhibiting thegrowth of or killing a fungus in a subject afflicted with a fungalinfection comprising administering to the subject an effective amount ofa compound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,        —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NHCOR₁₂, or —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   when R₁, R₂, R₃, R₄, R₅, R₆, R₈, R₁₁, R₁₂ are each —H, R₉ is        —Br, and —R₁₀ is —OCH₃, then R₇ is other than —CH₃; and    -   when R₁, R₂, R₃, R₄, R₅, R₈, R₉, R₁₁, and R₁₂ are each —H, and        —R₁₀ is OH, then R₆ and R₇ are other than —H and —CH₃ or —Br and        H, respectively,    -   or a pharmaceutically acceptable salt thereof, so as to thereby        inhibiting the growth of or kill the fungus in the subject        afflicted with the fungal infection.

In some embodiments, the method wherein at least one of R₈, R₉, R₁₀,R₁₁, and R₁₂ are other than —H. In some embodiments, the method whereinat least two of R₈, R₉, R₁₀, R₁₁, and R₁₂ are other than —H.

In some embodiments, the method wherein R₁ is —H or —CH₃; and R₂ is —Hor —CH₃. In some embodiments, the method wherein R₁ is —H; and R₂ is —H.

In some embodiments, the method wherein R₇ is C₂-C₁₂ alkyl or C₂-C₂₀alkyl. In some embodiments, the method

-   -   wherein R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br,        alkyl, —OH, —OR₁₃, or —NR₁₄R₁₅; and    -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H, —Br, alkyl,        —OH, or —OR₁₃,        -   wherein each occurrence of R₁₃, R₁₄, and R₁₅ is alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method

-   -   wherein R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br,        or alkyl; and    -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H, —Br, —OH,        or —OR₁₃,        -   wherein R₁₃ is alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method

-   -   wherein R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br,        or —CH₃; and    -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H, —Br, —OH,        or —OCH₃,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein the compound has the structure:

-   -   wherein    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NHCOR₁₂, or —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method

-   -   wherein R₈ and R₁₂ are each independently —H, —Br, or —NR₁₄R₁₅,    -   R₉, R₁₀, and R₁₁ are each independently —H, —Br, —OH, or —OR₁₃,        -   wherein each occurrence of R₁₃, R₁₄, and R₁₅ is alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method

-   -   wherein R₈ and R₁₂ are each independently —H, —Br, or —N(CH₃)₂,        R₉, R₁₀, and R₁₁ are each independently —H, —Br, —OH, or —OCH₃,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein the compound has the structure:

-   -   wherein    -   R₃, R₄, R₅, and R₆, and R₇ are each independently —H, halogen,        CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂,        —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method

-   -   wherein R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br,        or alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method

-   -   wherein R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br,        or —CH₃,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein the compound has the structure:

-   -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method further comprising administering aneffective amount of an anti-fungal agent.

In some embodiments, the method wherein the amount of the compound andthe amount of the anti-fungal agent when taken together is moreeffective to treat the subject than when the anti-fungal agent isadministered alone.

In some embodiments, the method wherein the amount of the compound andthe amount of the anti-fungal agent when taken together is effective toreduce a clinical symptom of the fungal infection in the subject.

In some embodiments, the method wherein the anti-fungal agent isfluconazole, amphotericin B, caspofungin, tunicamycin or aureobasidin A.

In some embodiments, the method wherein the fungal infection is causedby Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis,Stachybotrys or Mycrorales fungus.

In some embodiments, the method wherein the fungal infection is causedby Cryptococcus Neoformans.

In some embodiments, the method wherein the fungal infection isCryptococcus neoformans cryptococcosis.

In some embodiments, the method wherein the fungal infection isAspergillosis, Blastomycosis, Candidiasis, Coccidioidomycosis,Cryptococcus gattii cryptococcosis, Fungal Keratitis, Dermatophytes,Histoplasmosis, Mucormycosis, Pneumocystis pneumonia (PCP), orSporotrichosis.

In some embodiments, the method wherein the fungal infection is causedby Cryptococcus gattii, Candida albicans, Candida krusei, Candidaglabrata, Candida parapsilosis, Candida guilliermondii, Aspergillusfumigatus, Rhizopus oryzae, Rhizopus spp., Blastomyces dermatitis,Histoplasma capsulatum, Coccidioides spp., Paecilomyces variotii,Pneumocystis murina, Pneumocystis jiroveci, Histoplasma capsulatum,Aspergillus spp., or dimorphic fungi.

The present invention also provides a method of inhibiting the growth ofor killing a fungus in a subject afflicted with a fungal infectioncomprising administering to the subject an effective amount of acompound having the structure:

-   -   wherein    -   R₁ is —H, alkyl, alkenyl, or alkynyl;    -   R₂ is —H, alkyl, alkenyl, or alkynyl;    -   R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,        —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or        —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt or ester thereof, so as to        thereby inhibit the growth of or kill the fungus in the subject        afflicted with the fungal infection,    -   wherein the fungus in the subject afflicted with the fungal        infection is other than Cryptococcus neoformans.

In some embodiments, the method wherein at least one of R₈, R₉, R₁₀,R₁₁, and R₁₂ are other than —H. In some embodiments, the method whereinat least two of R₈, R₉, R₁₀, R₁₁, and R₁₂ are other than —H.

In some embodiments, the method wherein R₁ is —H or —CH₃; and R₂ is —Hor —CH₃. In some embodiments, the method wherein R₁ is —H; and R₂ is —H.

In some embodiments, the method wherein R₇ is C₂-C₁₂ alkyl or C₂-C₂₀alkyl.

In some embodiments, the method

-   -   wherein R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br,        alkyl, —OH, —OR₁₃, or —NR₁₄R₁₅; and    -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H, —Br, alkyl,        —OH, —OR₁₃ or NR₁₄R₁₅,        -   wherein each occurrence of R₁₃, R₁₄, and R₁₅ is alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method

-   -   wherein R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br,        or alkyl; and    -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H, —Br, —OH,        —OR₁₃, or NR₁₄R₁₅,        -   wherein each occurrence of R₁₃, R₁₄, and R₁₅ is alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method

-   -   wherein R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br,        or —CH₃; and    -   R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H, —Br, —OH,        —OCH₃, or —N(CH₃)₂,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein the compound has the structure:

-   -   wherein    -   R₈ and R₉ are each independently —H, halogen, —CN, —CF₃, —OCF₃,        —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,        —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃,        —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅;    -   R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃,        —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or        —CONR₁₄R₁₅; and    -   R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃,        —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH,        —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,        —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method

-   -   wherein R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H,        —Br, alkyl, —OH, —OR₁₃ or NR₁₄R₁₅,        -   wherein each occurrence of R₁₃, R₁₄, and R₁₅ is alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method

-   -   wherein R₈, R₉, R₁₀, R₁₁, and R₁₂ are each independently —H,        —Br, —OH, —OCH₃, or —N(CH₃)₂,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein the compound has the structure:

-   -   wherein    -   R₃, R₄, R₅, and R₆, and R₇ are each independently —H, halogen,        CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂,        —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅,        -   wherein each occurrence of R₁₃ is independently alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₄ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,        -   wherein each occurrence of R₁₅ is independently —H, alkyl,            alkenyl, alkynyl, aryl, or heteroaryl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method

-   -   wherein R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br,        or alkyl,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method

-   -   wherein R₃, R₄, R₅, R₆, and R₇ are each independently —H, —Br,        or —CH₃,    -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method wherein the compound has the structure:

-   -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the method further comprising administering aneffective amount of an anti-fungal agent.

In some embodiments, the method wherein the amount of the compound andthe amount of the anti-fungal agent when taken together is moreeffective to treat the subject than when the anti-fungal agent isadministered alone.

In some embodiments, the method wherein the amount of the compound andthe amount of the anti-fungal agent when taken together is effective toreduce a clinical symptom of the fungal infection in the subject.

In some embodiments, the method wherein the anti-fungal agent isfluconazole, amphotericin B, caspofungin, tunicamycin or aureobasidin A.

In some embodiments, the method wherein the fungal infection is causedby Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis,Stachybotrys or Mycrorales fungus.

In some embodiments, the method wherein the fungal infection isAspergillosis, Blastomycosis, Candidiasis, Coccidioidomycosis,Cryptococcus gattii cryptococcosis, Fungal Keratitis, Dermatophytes,Histoplasmosis, Mucormycosis, Pneumocystis pneumonia (PCP), orSporotrichosis.

In some embodiments, the method wherein the fungal infection is causedby Cryptococcus gattii, Candida albicans, Candida krusei, Candidaglabrata, Candida parapsilosis, Candida guilliermondii, Aspergillusfumigatus, Rhizopus oryzae, Rhizopus spp., Blastomyces dermatitis,Histoplasma capsulatum, Coccidioides spp., Paecilomyces variotii,Pneumocystis murina, Pneumocystis jiroveci, Histoplasma capsulatum, ordimorphic fungi.

In some embodiments, the fungal infection is an internal fungalinfection. In some embodiments, the fungal infection is an invasivefungal infection. In some embodiments, the fungal infection is a fungalinfection of the skin or lung. In some embodiments, the compound has afungistatic effect on the fungus. In some embodiments, the compound hasa fungicidal effect on the fungus. In some embodiments, the compound isadministered orally to the subject. In some embodiments, the compound isadministered topically to the subject. In some embodiments, the subjectis also afflicted with an immunodeficiency disorder. In someembodiments, the subject is also afflicted with human immunodeficiencyvirus (HIV).

In some embodiments, the antifungal agent is Amphotericin B, Candicidin,Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, Clotrimazole,Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole,Isoconazole, Ketoconazole, Luliconazole, Miconazole, Omoconazole,Oxiconazole, Sertaconazole, Sulconazole, Tioconazole, Albaconazole,Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Ravuconazole,Terconazole, Voriconazole, Abafungin, Amorolfin, Butenafine, Naftifine,Terbinafine, Anidulafungin, Caspofungin, Micafungin, Ciclopirox,Flucytosine, Griseofulvin, Haloprogin, Tolnaftate, or Undecylenic acid.In some embodiments, a pharmaceutical composition comprising a compoundof the present invention and an antifungal agent, and at least onepharmaceutically acceptable carrier for use in treating a fungalinfection.

In some embodiments, a pharmaceutical composition comprising an amountof the compound of the present invention for use in treating a subjectafflicted with a fungal infection as an add-on therapy or in combinationwith, or simultaneously, contemporaneously or concomitantly with ananti-fungal agent.

In some embodiments of any of the above methods or uses, the subject isa human. In some embodiments of any of the above methods or uses, thecompound and/or anti-fungal agent is orally administered to the subject.In some embodiments of any of the above methods or uses, the compoundand/or anti-fungal agent is topically administered to the subject.

In some embodiments, the fungus or fungal infection has developedresistance to one or more drugs. For example, a drug resistant fungalinfection may have developed drug-resistance to an azole antifungaldrug, a polyene antifungal drug and/or an echinocandin antifungal drug.

In some embodiments of any of the above methods or uses, the compoundtargets APL5, COS111, MKK1, and STE2 in the fungus.

In some embodiments of any of the above methods or uses, the compoundtargets at least one of APL5, COS111, MKK1, or STE2 in the fungus.

In some embodiments of any of the above methods or uses, the compounddisrupts vesicular transport mediate by APL5.

In some embodiments of any of the above methods or uses, the funguscarries non-mutated APL5, COS111, MKK1, and STE2.

In some embodiments of any of the above methods or uses, the funguscarries at least one of non-mutated APL5, COS111, MKK1, and STE2.

As used herein, a “symptom” associated with a fungal infection includesany clinical or laboratory manifestation associated with the fungalinfection and is not limited to what the subject can feel or observe.

As used herein, “treating”, e.g. of a fungal infection, encompassesinducing prevention, inhibition, regression, or stasis of the disease ora symptom or condition associated with the infection.

The compounds of the present invention include all hydrates, solvates,and complexes of the compounds used by this invention. If a chiralcenter or another form of an isomeric center is present in a compound ofthe present invention, all forms of such isomer or isomers, includingenantiomers and diastereomers, are intended to be covered herein.Compounds containing a chiral center may be used as a racemic mixture,an enantiomerically enriched mixture, or the racemic mixture may beseparated using well-known techniques and an individual enantiomer maybe used alone. The compounds described in the present invention are inracemic form or as individual enantiomers. The enantiomers can beseparated using known techniques, such as those described in Pure andApplied Chemistry 69, 1469-1474, (1997) IUPAC. In cases in whichcompounds have unsaturated carbon-carbon double bonds, both the cis (Z)and trans (E) isomers are within the scope of this invention.

The compounds of the subject invention may have spontaneous tautomericforms. In cases wherein compounds may exist in tautomeric forms, such asketo-enol tautomers, each tautomeric form is contemplated as beingincluded within this invention whether existing in equilibrium orpredominantly in one form.

In the compound structures depicted herein, hydrogen atoms are not shownfor carbon atoms having less than four bonds to non-hydrogen atoms.However, it is understood that enough hydrogen atoms exist on saidcarbon atoms to satisfy the octet rule.

This invention also provides isotopic variants of the compoundsdisclosed herein, including wherein the isotopic atom is ²H and/orwherein the isotopic atom ¹³C. Accordingly, in the compounds providedherein hydrogen can be enriched in the deuterium isotope. It is to beunderstood that the invention encompasses all such isotopic forms.

It is understood that the structures described in the embodiments of themethods hereinabove can be the same as the structures of the compoundsdescribed hereinabove.

It is understood that where a numerical range is recited herein, thepresent invention contemplates each integer between, and including, theupper and lower limits, unless otherwise stated.

Except where otherwise specified, if the structure of a compound of thisinvention includes an asymmetric carbon atom, it is understood that thecompound occurs as a racemate, racemic mixture, and isolated singleenantiomer. All such isomeric forms of these compounds are expresslyincluded in this invention. Except where otherwise specified, eachstereogenic carbon may be of the R or S configuration. It is to beunderstood accordingly that the isomers arising from such asymmetry(e.g., all enantiomers and diastereomers) are included within the scopeof this invention, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis, such as those described in“Enantiomers, Racemates and Resolutions” by J. Jacques, A. Collet and S.Wilen, Pub. John Wiley & Sons, N Y, 1981. For example, the resolutionmay be carried out by preparative chromatography on a chiral column.

The subject invention is also intended to include all isotopes of atomsoccurring on the compounds disclosed herein. Isotopes include thoseatoms having the same atomic number but different mass numbers. By wayof general example and without limitation, isotopes of hydrogen includetritium and deuterium. Isotopes of carbon include C-13 and C-14.

It will be noted that any notation of a carbon in structures throughoutthis application, when used without further notation, are intended torepresent all isotopes of carbon, such as ¹²C, ¹³C, or ¹⁴C. Furthermore,any compounds containing ¹³C or ¹⁴C may specifically have the structureof any of the compounds disclosed herein.

It will also be noted that any notation of a hydrogen in structuresthroughout this application, when used without further notation, areintended to represent all isotopes of hydrogen, such as ¹H, ²H, or ³H.Furthermore, any compounds containing ²H or ³H may specifically have thestructure of any of the compounds disclosed herein.

Isotopically-labeled compounds can generally be prepared by conventionaltechniques known to those skilled in the art using appropriateisotopically-labeled reagents in place of the non-labeled reagentsemployed.

In the compounds used in the method of the present invention, thesubstituents may be substituted or unsubstituted, unless specificallydefined otherwise.

In the compounds used in the method of the present invention, alkyl,heteroalkyl, monocycle, bicycle, aryl, heteroaryl and heterocycle groupscan be further substituted by replacing one or more hydrogen atoms withalternative non-hydrogen groups. These include, but are not limited to,halo, hydroxy, mercapto, amino, carboxy, cyano, carbamoyl andaminocarbonyl and aminothiocarbonyl.

It is understood that substituents and substitution patterns on thecompounds used in the method of the present invention can be selected byone of ordinary skill in the art to provide compounds that arechemically stable and that can be readily synthesized by techniquesknown in the art from readily available starting materials. If asubstituent is itself substituted with more than one group, it isunderstood that these multiple groups may be on the same carbon or ondifferent carbons, so long as a stable structure results.

In choosing the compounds used in the method of the present invention,one of ordinary skill in the art will recognize that the varioussubstituents, i.e. R₁, R₂, etc. are to be chosen in conformity withwell-known principles of chemical structure connectivity.

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms. Thus, C₁-C_(n) as in “C₁-C_(n) alkyl”is defined to include groups having 1, 2 . . . , n−1 or n carbons in alinear or branched arrangement, and specifically includes methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, isopropyl, isobutyl, sec-butyl andso on. An embodiment can be C₁-C₁₂ alkyl, C₂-C₁₂ alkyl, C₃-C₁₂ alkyl,C₄-C₁₂ alkyl and so on. “Alkoxy” represents an alkyl group as describedabove attached through an oxygen bridge.

The term “alkenyl” refers to a non-aromatic hydrocarbon radical,straight or branched, containing at least 1 carbon to carbon doublebond, and up to the maximum possible number of non-aromaticcarbon-carbon double bonds may be present. Thus, C₂-C_(n) alkenyl isdefined to include groups having 1, 2 . . . , n−1 or n carbons. Forexample, “C₂-C₆ alkenyl” means an alkenyl radical having 2, 3, 4, 5, or6 carbon atoms, and at least 1 carbon-carbon double bond, and up to, forexample, 3 carbon-carbon double bonds in the case of a C₆ alkenyl,respectively. Alkenyl groups include ethenyl, propenyl, butenyl andcyclohexenyl. As described above with respect to alkyl, the straight,branched or cyclic portion of the alkenyl group may contain double bondsand may be substituted if a substituted alkenyl group is indicated. Anembodiment can be C₂-C₁₂ alkenyl, C₃-C₁₂ alkenyl, C₄-C₁₂ alkenyl and soon.

The term “alkynyl” refers to a hydrocarbon radical straight or branched,containing at least 1 carbon to carbon triple bond, and up to themaximum possible number of non-aromatic carbon-carbon triple bonds maybe present. Thus, C₂-C_(n) alkynyl is defined to include groups having1, 2 . . . , n−1 or n carbons. For example, “C₂-C₆ alkynyl” means analkynyl radical having 2 or 3 carbon atoms, and 1 carbon-carbon triplebond, or having 4 or 5 carbon atoms, and up to 2 carbon-carbon triplebonds, or having 6 carbon atoms, and up to 3 carbon-carbon triple bonds.Alkynyl groups include ethynyl, propynyl and butynyl. As described abovewith respect to alkyl, the straight or branched portion of the alkynylgroup may contain triple bonds and may be substituted if a substitutedalkynyl group is indicated. An embodiment can be a C₂-C_(n) alkynyl. Anembodiment can be C₂-C₁₂ alkynyl, C₃-C₁₂ alkynyl, C₄-C₁₂ alkynyl and soon

“Alkylene”, “alkenylene” and “alkynylene” shall mean, respectively, adivalent alkane, alkene and alkyne radical, respectively. It isunderstood that an alkylene, alkenylene, and alkynylene may be straightor branched. An alkylene, alkenylene, and alkynylene may beunsubstituted or substituted.

As used herein, “heteroalkyl” includes both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms and at least 1 heteroatom within the chain or branch.

As used herein, “heterocycle” or “heterocyclyl” as used herein isintended to mean a 5- to 10-membered nonaromatic ring containing from 1to 4 heteroatoms selected from the group consisting of O, N and S, andincludes bicyclic groups. “Heterocyclyl” therefore includes, but is notlimited to the following: imidazolyl, piperazinyl, piperidinyl,pyrrolidinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl,dihydropiperidinyl, tetrahydrothiophenyl and the like. If theheterocycle contains a nitrogen, it is understood that the correspondingN-oxides thereof are also encompassed by this definition.

As herein, “cycloalkyl” shall mean cyclic rings of alkanes of three toeight total carbon atoms, or any number within this range (i.e.,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl orcyclooctyl).

As used herein, “monocycle” includes any stable polyatomic carbon ringof up to 10 atoms and may be unsubstituted or substituted. Examples ofsuch non-aromatic monocycle elements include but are not limited to:cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Examples of sucharomatic monocycle elements include but are not limited to: phenyl.

As used herein, “bicycle” includes any stable polyatomic carbon ring ofup to 10 atoms that is fused to a polyatomic carbon ring of up to 10atoms with each ring being independently unsubstituted or substituted.Examples of such non-aromatic bicycle elements include but are notlimited to: decahydronaphthalene. Examples of such aromatic bicycleelements include but are not limited to: naphthalene.

As used herein, “aryl” is intended to mean any stable monocyclic,bicyclic or polycyclic carbon ring of up to 10 atoms in each ring,wherein at least one ring is aromatic, and may be unsubstituted orsubstituted. Examples of such aryl elements include phenyl, p-toluenyl(4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, biphenyl,phenanthryl, anthryl or acenaphthyl. In cases where the aryl substituentis bicyclic and one ring is non-aromatic, it is understood thatattachment is via the aromatic ring.

As used herein, the term “polycyclic” refers to unsaturated or partiallyunsaturated multiple fused ring structures, which may be unsubstitutedor substituted.

The term “arylalkyl” refers to alkyl groups as described above whereinone or more bonds to hydrogen contained therein are replaced by a bondto an aryl group as described above. It is understood that an“arylalkyl” group is connected to a core molecule through a bond fromthe alkyl group and that the aryl group acts as a substituent on thealkyl group. Examples of arylalkyl moieties include, but are not limitedto, benzyl (phenylmethyl), p-trifluoromethylbenzyl(4-trifluoromethylphenylmethyl), 1-phenylethyl, 2-phenylethyl,3-phenylpropyl, 2-phenylpropyl and the like.

The term “heteroaryl”, as used herein, represents a stable monocyclic,bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein atleast one ring is aromatic and contains from 1 to 4 heteroatoms selectedfrom the group consisting of O, N and S. Bicyclic aromatic heteroarylgroups include phenyl, pyridine, pyrimidine or pyridizine rings that are(a) fused to a 6-membered aromatic (unsaturated) heterocyclic ringhaving one nitrogen atom; (b) fused to a 5- or 6-membered aromatic(unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused toa 5-membered aromatic (unsaturated) heterocyclic ring having onenitrogen atom together with either one oxygen or one sulfur atom; or (d)fused to a 5-membered aromatic (unsaturated) heterocyclic ring havingone heteroatom selected from O, N or S. Heteroaryl groups within thescope of this definition include but are not limited to:benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl,benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl,cinnolinyl, furanyl, indolinyl, indolyl, indolazinyl, indazolyl,isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl,naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline,oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl,pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl,quinoxalinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl,thienyl, triazolyl, azetidinyl, aziridinyl, 1,4-dioxanyl,hexahydroazepinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl,dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl,dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, acridinyl,carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl,benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl,furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl,oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl,pyrimidinyl, pyrrolyl, tetra-hydroquinoline. In cases where theheteroaryl substituent is bicyclic and one ring is non-aromatic orcontains no heteroatoms, it is understood that attachment is via thearomatic ring or via the heteroatom containing ring, respectively. Ifthe heteroaryl contains nitrogen atoms, it is understood that thecorresponding N-oxides thereof are also encompassed by this definition.

The term “alkylheteroaryl” refers to alkyl groups as described abovewherein one or more bonds to hydrogen contained therein are replaced bya bond to an heteroaryl group as described above. It is understood thatan “alkylheteroaryl” group is connected to a core molecule through abond from the alkyl group and that the heteroaryl group acts as asubstituent on the alkyl group. Examples of alkylheteroaryl moietiesinclude, but are not limited to, —CH₂—(C₅H₄N), —CH₂—CH₂—(C₅H₄N) and thelike.

The term “heterocycle” or “heterocyclyl” refers to a mono- orpoly-cyclic ring system which can be saturated or contains one or moredegrees of unsaturation and contains one or more heteroatoms. Preferredheteroatoms include N, O, and/or S, including N-oxides, sulfur oxides,and dioxides. Preferably the ring is three to ten-membered and is eithersaturated or has one or more degrees of unsaturation. The heterocyclemay be unsubstituted or substituted, with multiple degrees ofsubstitution being allowed. Such rings may be optionally fused to one ormore of another “heterocyclic” ring(s), heteroaryl ring(s), arylring(s), or cycloalkyl ring(s). Examples of heterocycles include, butare not limited to, tetrahydrofuran, pyran, 1,4-dioxane, 1,3-dioxane,piperidine, piperazine, pyrrolidine, morpholine, thiomorpholine,tetrahydrothiopyran, tetrahydrothiophene, 1,3-oxathiolane, and the like.

The alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclylsubstituents may be substituted or unsubstituted, unless specificallydefined otherwise. In the compounds of the present invention, alkyl,alkenyl, alkynyl, aryl, heterocyclyl and heteroaryl groups can befurther substituted by replacing one or more hydrogen atoms withalternative non-hydrogen groups. These include, but are not limited to,halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.

As used herein, the term “halogen” refers to F, Cl, Br, and I.

The terms “substitution”, “substituted” and “substituent” refer to afunctional group as described above in which one or more bonds to ahydrogen atom contained therein are replaced by a bond to non-hydrogenor non-carbon atoms, provided that normal valencies are maintained andthat the substitution results in a stable compound. Substituted groupsalso include groups in which one or more bonds to a carbon(s) orhydrogen(s) atom are replaced by one or more bonds, including double ortriple bonds, to a heteroatom. Examples of substituent groups includethe functional groups described above, and halogens (i.e., F, Cl, Br,and I); alkyl groups, such as methyl, ethyl, n-propyl, isopropryl,n-butyl, tert-butyl, and trifluoromethyl; hydroxyl; alkoxy groups, suchas methoxy, ethoxy, n-propoxy, and isopropoxy; aryloxy groups, such asphenoxy; arylalkyloxy, such as benzyloxy (phenylmethoxy) andp-trifluoromethylbenzyloxy (4-trifluoromethylphenylmethoxy);heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl,methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto;sulfanyl groups, such as methylsulfanyl, ethylsulfanyl andpropylsulfanyl; cyano; amino groups, such as amino, methylamino,dimethylamino, ethylamino, and diethylamino; and carboxyl. Wheremultiple substituent moieties are disclosed or claimed, the substitutedcompound can be independently substituted by one or more of thedisclosed or claimed substituent moieties, singly or pluraly. Byindependently substituted, it is meant that the (two or more)substituents can be the same or different.

It is understood that substituents and substitution patterns on thecompounds of the instant invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art, as wellas those methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.

In choosing the compounds of the present invention, one of ordinaryskill in the art will recognize that the various substituents, i.e. R₁,R₂, etc. are to be chosen in conformity with well-known principles ofchemical structure connectivity.

The various R groups attached to the aromatic rings of the compoundsdisclosed herein may be added to the rings by standard procedures, forexample those set forth in Advanced Organic Chemistry: Part B: Reactionand Synthesis, Francis Carey and Richard Sundberg, (Springer) 5th ed.Edition. (2007), the content of which is hereby incorporated byreference.

The compounds used in the method of the present invention may beprepared by techniques well known in organic synthesis and familiar to apractitioner ordinarily skilled in the art. However, these may not bethe only means by which to synthesize or obtain the desired compounds.

The compounds used in the method of the present invention may beprepared by techniques described in Vogel's Textbook of PracticalOrganic Chemistry, A. I. Vogel, A. R. Tatchell, B. S. Furnis, A. J.Hannaford, P. W. G. Smith, (Prentice Hall) 5^(th) Edition (1996),March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5^(th)Edition (2007), and references therein, which are incorporated byreference herein. However, these may not be the only means by which tosynthesize or obtain the desired compounds.

Another aspect of the invention comprises a compound used in the methodof the present invention as a pharmaceutical composition.

In some embodiments, a pharmaceutical composition comprising thecompound of the present invention and a pharmaceutically acceptablecarrier.

As used herein, the term “pharmaceutically active agent” means anysubstance or compound suitable for administration to a subject andfurnishes biological activity or other direct effect in the treatment,cure, mitigation, diagnosis, or prevention of disease, or affects thestructure or any function of the subject. Pharmaceutically active agentsinclude, but are not limited to, substances and compounds described inthe Physicians' Desk Reference (PDR Network, LLC; 64th edition; Nov. 15,2009) and “Approved Drug Products with Therapeutic EquivalenceEvaluations” (U.S. Department Of Health And Human Services, 30^(th)edition, 2010), which are hereby incorporated by reference.Pharmaceutically active agents which have pendant carboxylic acid groupsmay be modified in accordance with the present invention using standardesterification reactions and methods readily available and known tothose having ordinary skill in the art of chemical synthesis. Where apharmaceutically active agent does not possess a carboxylic acid group,the ordinarily skilled artisan will be able to design and incorporate acarboxylic acid group into the pharmaceutically active agent whereesterification may subsequently be carried out so long as themodification does not interfere with the pharmaceutically active agent'sbiological activity or effect.

The compounds used in the method of the present invention may be in asalt form. As used herein, a “salt” is a salt of the instant compoundswhich has been modified by making acid or base salts of the compounds.In the case of compounds used to treat an infection or disease caused bya pathogen, the salt is pharmaceutically acceptable. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as phenols. The salts can bemade using an organic or inorganic acid. Such acid salts are chlorides,bromides, sulfates, nitrates, phosphates, sulfonates, formates,tartrates, maleates, malates, citrates, benzoates, salicylates,ascorbates, and the like. Phenolate salts are the alkaline earth metalsalts, sodium, potassium or lithium. The term “pharmaceuticallyacceptable salt” in this respect, refers to the relatively non-toxic,inorganic and organic acid or base addition salts of compounds of thepresent invention. These salts can be prepared in situ during the finalisolation and purification of the compounds of the invention, or byseparately reacting a purified compound of the invention in its freebase or free acid form with a suitable organic or inorganic acid orbase, and isolating the salt thus formed. Representative salts includethe hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like. (See, e.g., Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).

The compounds of the present invention may also form salts with basicamino acids such a lysine, arginine, etc. and with basic sugars such asN-methylglucamine, 2-amino-2-deoxyglucose, etc. and any otherphysiologically non-toxic basic substance.

As used herein, “administering” an agent may be performed using any ofthe various methods or delivery systems well known to those skilled inthe art. The administering can be performed, for example, orally,parenterally, intraperitoneally, intravenously, intraarterially,transdermally, sublingually, intramuscularly, rectally, transbuccally,intranasally, liposomally, via inhalation, vaginally, intraoccularly,via local delivery, subcutaneously, intraadiposally, intraarticularly,intrathecally, into a cerebral ventricle, intraventicularly,intratumorally, into cerebral parenchyma or intraparenchchymally.

The compounds used in the method of the present invention may beadministered in various forms, including those detailed herein. Thetreatment with the compound may be a component of a combination therapyor an adjunct therapy, i.e. the subject or patient in need of the drugis treated or given another drug for the disease in conjunction with oneor more of the instant compounds. This combination therapy can besequential therapy where the patient is treated first with one drug andthen the other or the two drugs are given simultaneously. These can beadministered independently by the same route or by two or more differentroutes of administration depending on the dosage forms employed.

As used herein, a “pharmaceutically acceptable carrier” is apharmaceutically acceptable solvent, suspending agent or vehicle, fordelivering the instant compounds to the animal or human. The carrier maybe liquid or solid and is selected with the planned manner ofadministration in mind. Liposomes are also a pharmaceutically acceptablecarrier as are slow-release vehicles.

The dosage of the compounds administered in treatment will varydepending upon factors such as the pharmacodynamic characteristics of aspecific chemotherapeutic agent and its mode and route ofadministration; the age, sex, metabolic rate, absorptive efficiency,health and weight of the recipient; the nature and extent of thesymptoms; the kind of concurrent treatment being administered; thefrequency of treatment with; and the desired therapeutic effect.

A dosage unit of the compounds used in the method of the presentinvention may comprise a single compound or mixtures thereof withadditional antitumor agents. The compounds can be administered in oraldosage forms as tablets, capsules, pills, powders, granules, elixirs,tinctures, suspensions, syrups, and emulsions. The compounds may also beadministered in intravenous (bolus or infusion), intraperitoneal,subcutaneous, or intramuscular form, or introduced directly, e.g. byinjection, topical application, or other methods, into or topically ontoa site of disease or lesion, all using dosage forms well known to thoseof ordinary skill in the pharmaceutical arts.

The compounds used in the method of the present invention can beadministered in admixture with suitable pharmaceutical diluents,extenders, excipients, or in carriers such as the novel programmablesustained-release multi-compartmental nanospheres (collectively referredto herein as a pharmaceutically acceptable carrier) suitably selectedwith respect to the intended form of administration and as consistentwith conventional pharmaceutical practices. The unit will be in a formsuitable for oral, nasal, rectal, topical, intravenous or directinjection or parenteral administration. The compounds can beadministered alone or mixed with a pharmaceutically acceptable carrier.This carrier can be a solid or liquid, and the type of carrier isgenerally chosen based on the type of administration being used. Theactive agent can be co-administered in the form of a tablet or capsule,liposome, as an agglomerated powder or in a liquid form. Examples ofsuitable solid carriers include lactose, sucrose, gelatin and agar.Capsule or tablets can be easily formulated and can be made easy toswallow or chew; other solid forms include granules, and bulk powders.Tablets may contain suitable binders, lubricants, diluents,disintegrating agents, coloring agents, flavoring agents, flow-inducingagents, and melting agents. Examples of suitable liquid dosage formsinclude solutions or suspensions in water, pharmaceutically acceptablefats and oils, alcohols or other organic solvents, including esters,emulsions, syrups or elixirs, suspensions, solutions and/or suspensionsreconstituted from non-effervescent granules and effervescentpreparations reconstituted from effervescent granules. Such liquiddosage forms may contain, for example, suitable solvents, preservatives,emulsifying agents, suspending agents, diluents, sweeteners, thickeners,and melting agents. Oral dosage forms optionally contain flavorants andcoloring agents. Parenteral and intravenous forms may also includeminerals and other materials to make them compatible with the type ofinjection or delivery system chosen.

Techniques and compositions for making dosage forms useful in thepresent invention are described in the following references: 7 ModernPharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979);Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel,Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976);Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company,Easton, Pa., 1985); Advances in Pharmaceutical Sciences (DavidGanderton, Trevor Jones, Eds., 1992); Advances in PharmaceuticalSciences Vol. 7. (David Ganderton, Trevor Jones, James McGinity, Eds.,1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugsand the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989);Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs andthe Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); DrugDelivery to the Gastrointestinal Tract (Ellis Horwood Books in theBiological Sciences. Series in Pharmaceutical Technology; J. G. Hardy,S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and thePharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T.Rhodes, Eds.). All of the aforementioned publications are incorporatedby reference herein.

Tablets may contain suitable binders, lubricants, disintegrating agents,coloring agents, flavoring agents, flow-inducing agents, and meltingagents. For instance, for oral administration in the dosage unit form ofa tablet or capsule, the active drug component can be combined with anoral, non-toxic, pharmaceutically acceptable, inert carrier such aslactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose,magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol,sorbitol and the like. Suitable binders include starch, gelatin, naturalsugars such as glucose or beta-lactose, corn sweeteners, natural andsynthetic gums such as acacia, tragacanth, or sodium alginate,carboxymethylcellulose, polyethylene glycol, waxes, and the like.Lubricants used in these dosage forms include sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate, sodiumchloride, and the like. Disintegrators include, without limitation,starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

The compounds used in the method of the present invention may also beadministered in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles, and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids suchas lecithin, sphingomyelin, proteolipids, protein-encapsulated vesiclesor from cholesterol, stearylamine, or phosphatidylcholines. Thecompounds may be administered as components of tissue-targetedemulsions.

The compounds used in the method of the present invention may also becoupled to soluble polymers as targetable drug carriers or as a prodrug.Such polymers include polyvinylpyrrolidone, pyran copolymer,polyhydroxylpropylmethacrylamide-phenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compounds may becoupled to a class of biodegradable polymers useful in achievingcontrolled release of a drug, for example, polylactic acid, polyglycolicacid, copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacylates, and crosslinked or amphipathicblock copolymers of hydrogels.

Gelatin capsules may contain the active ingredient compounds andpowdered carriers, such as lactose, starch, cellulose derivatives,magnesium stearate, stearic acid, and the like. Similar diluents can beused to make compressed tablets. Both tablets and capsules can bemanufactured as immediate release products or as sustained releaseproducts to provide for continuous release of medication over a periodof hours. Compressed tablets can be sugar-coated or film-coated to maskany unpleasant taste and protect the tablet from the atmosphere, orenteric coated for selective disintegration in the gastrointestinaltract.

For oral administration in liquid dosage form, the oral drug componentsare combined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Examples ofsuitable liquid dosage forms include solutions or suspensions in water,pharmaceutically acceptable fats and oils, alcohols or other organicsolvents, including esters, emulsions, syrups or elixirs, suspensions,solutions and/or suspensions reconstituted from non-effervescentgranules and effervescent preparations reconstituted from effervescentgranules. Such liquid dosage forms may contain, for example, suitablesolvents, preservatives, emulsifying agents, suspending agents,diluents, sweeteners, thickeners, and melting agents.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance. In general, water, asuitableoil, saline, aqueous dextrose (glucose), and related sugar solutions andglycols such as propylene glycol or polyethylene glycols are suitablecarriers for parenteral solutions. Solutions for parenteraladministration preferably contain a water soluble salt of the activeingredient, suitable stabilizing agents, and if necessary, buffersubstances. Antioxidizing agents such as sodium bisulfite, sodiumsulfite, or ascorbic acid, either alone or combined, are suitablestabilizing agents. Also used are citric acid and its salts and sodiumEDTA. In addition, parenteral solutions can contain preservatives, suchas benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field.

The compounds used in the method of the present invention may also beadministered in intranasal form via use of suitable intranasal vehicles,or via transdermal routes, using those forms of transdermal skin patcheswell known to those of ordinary skill in that art. To be administered inthe form of a transdermal delivery system, the dosage administrationwill generally be continuous rather than intermittent throughout thedosage regimen.

Parenteral and intravenous forms may also include minerals and othermaterials such as solutol and/or ethanol to make them compatible withthe type of injection or delivery system chosen.

The compounds and compositions of the present invention can beadministered in oral dosage forms as tablets, capsules, pills, powders,granules, elixirs, tinctures, suspensions, syrups, and emulsions. Thecompounds may also be administered in intravenous (bolus or infusion),intraperitoneal, subcutaneous, or intramuscular form, or introduceddirectly, e.g. by topical administration, injection or other methods, tothe afflicted area, such as a wound, including ulcers of the skin, allusing dosage forms well known to those of ordinary skill in thepharmaceutical arts.

Specific examples of pharmaceutically acceptable carriers and excipientsthat may be used to formulate oral dosage forms of the present inventionare described in U.S. Pat. No. 3,903,297 to Robert, issued Sep. 2, 1975.Techniques and compositions for making dosage forms useful in thepresent invention are described-in the following references: 7 ModernPharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979);Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel,Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976);Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company,Easton, Pa., 1985); Advances in Pharmaceutical Sciences (DavidGanderton, Trevor Jones, Eds., 1992); Advances in PharmaceuticalSciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds.,1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugsand the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989);Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs andthe Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); DrugDelivery to the Gastrointestinal Tract (Ellis Horwood Books in theBiological Sciences. Series in Pharmaceutical Technology; J. G. Hardy,S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and thePharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T.Rhodes, Eds.). All of the aforementioned publications are incorporatedby reference herein.

The active ingredient can be administered orally in solid dosage forms,such as capsules, tablets, powders, and chewing gum; or in liquid dosageforms, such as elixirs, syrups, and suspensions, including, but notlimited to, mouthwash and toothpaste. It can also be administeredparentally, in sterile liquid dosage forms.

Solid dosage forms, such as capsules and tablets, may be enteric-coatedto prevent release of the active ingredient compounds before they reachthe small intestine. Materials that may be used as enteric coatingsinclude, but are not limited to, sugars, fatty acids, proteinaceoussubstances such as gelatin, waxes, shellac, cellulose acetate phthalate(CAP), methyl acrylate-methacrylic acid copolymers, cellulose acetatesuccinate, hydroxy propyl methyl cellulose phthalate, hydroxy propylmethyl cellulose acetate succinate (hypromellose acetate succinate),polyvinyl acetate phthalate (PVAP), and methyl methacrylate-methacrylicacid copolymers.

The compounds and compositions of the invention can be coated ontostents for temporary or permanent implantation into the cardiovascularsystem of a subject.

Variations on those general synthetic methods will be readily apparentto those of ordinary skill in the art and are deemed to be within thescope of the present invention.

Each embodiment disclosed herein is contemplated as being applicable toeach of the other disclosed embodiments. Thus, all combinations of thevarious elements described herein are within the scope of the invention.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

Experimental Details Materials and Methods Strains, Media and Reagents

A series of fungal clinical isolates and reference strains were used inthis study. This includes Cryptococcus neoformans, Cryptococcus gattii,Candida albicans, Candida krusei, Candida glabrata, Candidaparapsilosis, Candida guilliermondii, Aspergillus fumigatus, Rhizopusoryzae, Blastomyces dermatitis, Histoplasma capsulatum, Coccidioidesspp. Paecilomyces variotii, Pneumocystis murina, and, Pneumocystisjiroveci (see Table 1 and Table 2). Escherichia coli DH5-α andPseudomonas aeruginosa were also used. Yeast Peptone Dextrose (YPD),Yeast Nitrogen Base (YNB), Luria Bertani (LB), Roswell Park MemorialInstitute (RPMI) or Dulbecco Modified Eagle Medium (DMEM) were purchasedfrom Invitrogen Life Technologies and used as described. Fluconazole,Amphotericin B, Dexamethasone, Cyclophosphamide, Tunicamycin werepurchased from Sigma-Aldrich, St Louis, Mo. Caspofungin and Posaconazolewere obtained from Merck, Rahway, N.J. Voriconazole was obtained fromPfizer, Rey Brook, N.Y.N′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and3-bromo-N′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (1) wereobtained from ChemBridge, San Diego, Calif. Cryptococcal capsularantibody mAb 18B7 was a gift from Casadevall's Lab.

Library Screening

The DIVERSet™-CL library was obtained from ChemBridge and contained 10mM compound(s) per well in 100% DMSO in 96 well plate format. In eachwell, 10 compounds were mixed together. The compounds were first dilutedto 1 mM each (1:10 dilution, 10% DMSO) with yeast nitrogen base (YNB)medium buffered with HEPES at pH 7.4 containing 2% glucose andsubsequently diluted to 300 μM (1:3.3 dilution) with the same medium (3%DMSO). Then, 100 μL of this solution was placed into each well of 96well plates and stored at −20° C. until use. Then, 4×10⁴ C. neoformanscells in 100 μL of YNB medium buffered at pH 7.4 with HEPES were addedto each well. Thus, final concentration of the tested drugs was 150 μMin YNB medium containing 1.5% DMSO. The plates were incubated at 37° C.in the presence of 5% CO₂ for 48 hrs. Optical density at 495 nm wasrecorded using the Multi-mode microplate reader (FilterMax 5, MolecularDevice, Sunnyvale, Calif.) and cocktail compounds in wells showing anOD<80% compared to the OD in the control well (1.5% DMSO but no drug)were selected for further studies. Schematic of the high throughputscreening is summarized in FIG. 8.

Synthesis of BHBM Derivatives

The benzaldehyde (1 mmol, 2 ml ethanol) and the benzohydrazide (1 mmolin 2 ml hot ethanol) were combined. All products—except 3 and7—crystallized within seconds. After 30 minutes at room temperature theproduct was collected by filtration (Yield: 80 to 95%). Homogeneity ofthe product was confirmed by thin layer chromatography (TLC) onsilicagel F₂₅₄ (Merck KGaA, Darmstadt, Germany) in two different solventsystems benzene/acetic acid 9:1 v/v and hexane/ethylacetate 1:3 v/v. Ifimpurities were present the product was recrystallized from ethanol. Forthe synthesis of compounds 3 and 7 the reaction was conducted asdescribed above. After 24 h at 4° C. the solvent was completelyevaporated and the product crystallized from ethylacetate (Yields:3=72.9%, 7=67.3%). Products were analyzed by TLC as described above.

In Vivo Labeling with Tritiated Palmitate (³H Palmitate)

Labeling fungal cells. C. neoformans cells were grown in YNB (pH 7.4) at37° C. in presence of 5% CO₂ for 16 hrs. Cells were centrifuged for 10min at 3,000 rpm at room temperature. Supernatant was removed and thecell pellet was suspended and counted. Next, 900 μL containing 5×10⁸ C.neoformans cells were placed into a 15 ml round bottom Corningcentrifuge tube. Then, 100 μL of different concentrations of BHBM or 1diluted in YNB containing 0.1% DMSO was added resulting in finalconcentrations of 0.25, 1 and 4 μg/ml, or 0.075, 0.3, 1.2 μg/ml,respectively. Tubes were incubated at in a shaker incubator at 225 rpmat 37° C. in the presence of 5% CO₂ for 4 hours. Then, 30 μCi/ml of ³Hpalmitate (PerkinElmer, Waltham, Mass.) was added to the culture andincubated for additional 2 hours. Cells without the drug were includedas negative control. The cells were then pelleted and washed once withdistilled sterile water and suspended in 1.5 ml of Mandala lipidextraction buffer. The lipids were extracted by the methods of Mandala,(Mandala, S. M. et al. 1997) and Bligh and Dyer followed by methanolicbased-hydrolysis as previously described (Bligh, E. G. & Dyer, W. J.1959). The tube was flushed with nitrogen gas and the samples dried in aSPD210 SpeedVac system (ThermoFisher Scientific, Waltham, Mass. Thedried lipids were resuspended in 30 μL of 1:1 methanol:chloroform andloaded on thin layered chromatography (TLC) silica gel 60 (EDMMillipore, Billerica, Mass.). Glucosylceramide (GlcCer) standard fromsoybean (Avanti Polar Lipids, Alabaster, Ala.) was added in a separatelane as control. The sample was resolved in a tank containing achloroform:methanol:water (65:25:4) as the mobile phase. The TLC plateswere then dried, exposed to iodine fume for the identification of theGlcCer standard band, which was marked. The TLC plate was then enhancedby spraying with ENHENCER (PerkinElmer) exposed to X-Ray film at −80° C.for 72 hours and the film was developed.

Labeling Mammalian Cells.

The murine macrophage cell line J774.16 was maintained in DulbeccoMinimum Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and1% Pen-strep by regular seeding. Cell at a density of 5×10⁶ cells/ml ofpassage 8 were cultured in a 6 well culture plate for 14 hours toachieve adherence. BHBM or 1 at the same concentrations used for fungalcells (see above) were added to the plate for 4 hours. Then, 30 μCi/mLof ³H palmitic acid was added and the plate was further incubated for 2hrs. Labeled J774.16 but untreated cells were included as control. Thecells were harvested by the addition of 0.05% trypsin-EDTA and scrapingwith cell scrapper, and washed once with PBS and dissolved in 2 mlmethanol and 1 ml chloroform. Lipids were extracted by the method ofBligh and Dyer followed by base hydrolysis. The samples were flushedwith nitrogen and dried in SpeedVac. Dried lipids were suspended in 30μL of 1:1 methanol:chloroform and loaded on a TLC plate with GlcCer asstandard.

In Vitro Susceptibility Testing

Minimal inhibitory concentration (MIC) was determined following themethods of the Clinical Laboratory Standards Institutes (CLSI) withmodifications. MIC studies used either RPMI or YNB medium (pH 7.0, 0.2%glucose) buffered with HEPES. HEPES was used instead of MOPS becauseMOPS totally inhibits the activity of BHBM or of its derivative 1. BHBMor 1 was serially diluted from 32 to 0.03 μg/ml or 19 to 0.02 μg/mlrespectively in a 96 well plate with the respective medium. The yeastinoculum was prepared as described in the CLSI protocol M27-A3guidelines. Plates were incubated at 37° C. and in the presence of 5%CO₂ for 24-96 hours (see Table 2). Against all fungal isolates used inthe initial susceptibility screen, the MICs were determined as thelowest concentration of the drug that inhibited 50% of growth comparedto the control. MIC80 and MIC100, whose drug concentrations inhibited80% and 100% growth compared to the control respectively, were alsodetermined. For antibacterial activity, E. coli DH5a and P. aeruginosaPA14 were grown overnight in Luria Bertani (LB) broth at 30° C. Thecells were washed with PBS and counted. Then, 300 μL from 2×10⁸ cells/mLwas spreaded onto LB agar plate using a hockey stick glass spreader. Theplate was dried and wells were punched out using a cut tips. Fiftymicroliters of different drug concentration was added to the well. Theplate was then incubated at 30° C. for 24 hours.

In Vitro Testing Against P. murina and P. jiroveci

Cryopreserved and characterized P. carinii isolated from rat lung tissue(Pc 08-4#45) was distributed into triplicate wells of 48-well plateswith a final volume of 500 μL and a final concentration of 5×10⁷nuclei/ml. Control dilutions were added and incubated at 37° C. At 24,48, and 72 hours, 10% of the well volume was removed and the ATP contentwas measured using Perkin Elmer ATP-liteM luciferin-luciferase assay.The luminescence generated by the ATP content of the samples wasmeasured by a spectrophotometer (PolarStar Optima BMG, Ortenberg,Germany). A sample of each group was examined microscopically on thefinal assay day of the assay to rule out the presence of bacteriacontamination.

In Vitro Killing Assay

From an overnight culture, C. neoformans cells were washed in PBS,resuspended in YNB buffered with HEPES at pH 7.4. Cells were counted and2×10⁴ cells were incubated with either 1, 2 or 4 μg/ml of either BHBM or1 in a final volume of 10 ml. Tubes were then incubated at 37° C. in thepresence of 5% CO₂ on a rotary shaker at 200 rpm. At the illustratedtime points, aliquots were taken and diluted and 100 μL was plated ontoyeast peptone dextrose (YPD) plates. YPD plates were incubated in a 30°C. incubator and, after 72 hours, colony forming units (CFU) werecounted and recorded.

Intracellular Effect of BHBM

To assess whether BHBM will be effective against intracellular C.neoformans, we first incubated J774.16 macrophages with C. neoformanscells at a 1:20 ratio in presence of opsonins (complement and antibodymAb 18B7 against the cryptococcal capsular antigen). After 2 hours ofincubation, about 60-80% of macrophages have at least one C. neoformanscell internalized. At this time, wells were washed to removeextracellular fungal cells and fresh DMEM medium without serum andwithout mAb 18B7 but containing different concentrations of BHBM wasadded. Plates were incubated at 37° C. and 5% CO₂. At selected timepoints, 0, 6, 12 and 24 hours, extracellular cells were collected bywashing and plated onto YPD for CFU counting of extracellular cells.Then, macrophages containing C. neoformans were lysed, collected andserial dilutions were plated onto YPD for CFU counting of intracellularfungal cells.

Synergistic Assay

Synergistic activity was assayed by calculating the fractionalinhibitory index (FIC) as previously described (Del Poeta, M. et al.2000). Briefly, in a 96 well plate, drug A (either BHBM or 1) wasserially diluted from 16 to 0.015 μg/ml (11 dilutions) whereas drug B(either Fluconazole, Amphotericin B, Caspofungin, or Tunicamycin) wasserially diluted from 12 to 0.19 μg/ml, 5 to 0.078 μg/ml, 70 to 1.09μg/ml, and 6 to 0.09 μg/ml (7 dilutions), respectively. The FIC wasdefined as: [MIC combined/MIC Drug A alone]+[MIC combined/MIC Dug Balone].

Resistance Assay

To see whether incubation with the drugs will induce resistance, C.neoformans cells were passaged daily in sub-MIC drug concentrations.Briefly, from an overnight culture, C. neoformans cells were washed withPBS, resuspended in YNB buffered with HEPES at pH 7.4 and counted. Then,10⁶ cells were incubated with 0.5, 0.25 or 0.125 μg/ml of BHBM or 0.15,0.075 and 0.037 μg/ml of 1 in 1 ml final volume. Tubes without the drugserved as negative control. Tubes with Fluconazole (0.5, 1 and 2 μg/ml)served as positive control. The cells were grown at 37° C. in thepresence on 5% CO₂ on a rotary shaker at 200 rpm. Every 24 hours, thecells were pelleted by centrifugation, washed with PBS, and resuspendedin YNB, and 10⁶ cells were transferred into a fresh drug tube andincubated as above. These daily passages were continued for 15 days.Cell aliquots were collected on day 0 (before any drug exposure), 5, 10,15, and MIC was determined using the microbroth dilution assay asdescribed above.

Animal Studies for Cryptococcosis

For survival studies, 4-week old CBA/J female mice (Jackson Laboratory,Bar Harbor, Me.) were used. Ten mice per treatment or control group wereused. Mice were infected by nasal inoculation of 20 μL containing 5×10⁵cells of C. neoformans H99 strain. Treated mice received anintraperitoneal injection of 1.2 mg/kg/day of either BHBM or 1 in 100 μLfinal volume of PBS containing 0.4% DMSO. Untreated mice, received 100μL of PBS/0.4% DMSO. Mice were feed ad-libitum and monitored closely forsign of discomfort and meningitis. Mice showing abnormal gait,lethargic, tremor, significant loss of body weight or inability to reachwater or food were sacrificed and survival counted from that day. At theend of the survival study, tissue burden culture was performed in micethat survived the infection. Mice were sacrificed and their organs wereextracted, and homogenized in 10 ml sterile PBS using a homogenizer(Stomacher80, Cole-Parmer, Vernon Hills, Ill.). Organ homogenates wereserially diluted 1:10 in PBS and 100 μL was plated on YPD agar platesand incubated at 30° C. for 72 hours for CFU count. For histopathology,extracted organs were fixed in 10% formalin before paraffin sectioningand staining with either Hematoxylin-Eosin or Mucicarmine. Images weretaken at 40× in a Zeiss Axio Observer in brightfield mode.

Animal Studies for Pneumocystosis

For survival studies, C3H/HeN mice ordered from the National CancerInstitute (Bethesda, Md.) were used. Mice were infected with P. murinapneumonia through exposure to mice with a fulminant P. murina infection(seed mice). These mice were immune suppressed by the addition ofdexamethasone at 4 mg/liter to the drinking water. Sulfuric acid at 1ml/liter was also added to the drinking water for disinfection. The seedmice are rotated within the cages for 2 weeks and then removed. Afterthe mice had developed a moderate infection level (approximately 5weeks), they were divided into a negative control group (controlsteroid), positive control group (trimethoprim/sulfamethoxazole) andtreatment groups (BHBM or 1). Twelve mice were used in each group. BHBMor 1 were administered intraperitoneally or by oral gavage on amg/kg/day basis for up to 3 weeks. The dose, route, and frequency ofadministration varied depending on the agent being tested. At the end ofthe treatment, mice were sacrificed and processed for analysis. Slideswere made from the lung homogenates at different dilutions and stainedwith Diff-Quik to quantify the trophic forms and Cresyl Echt violet toquantify the asci. Additional group of mice were selectively depleted oftheir CD4+ lymphocytes by antibody treatment with 300 μg of GK 1.5antibody (Biovest International, Minneapolis, Minn.) administeredintraperitoneally 3 times on days 1, 3, and 7. After this initialtreatment, the mice were infected by exposure to P. murina infectedmice. Mice then were treated with 100 μg of GK 1.5 antibodyintraperitoneally once a week for 6 weeks. Mice were then treated with1.25 or 12.5 mg/kg/day of 1 for 14 days while continuing the GK1.5treatment. Control mice received vehicle.

Animal Studies for Candidiasis

For survival studies, 8-week old CBA/J female mice (Jackson Laboratory)were used. Eight mice per treatment or control group were used. Micewere infected by intravenous inoculation of 100 μL containing 1×10⁵cells of Candida albicans SC-5314 strain. Treated mice received anintraperitoneal injection of 1.2 mg/kg/day of either BHBM or 1 in 100 μLfinal volume of PBS containing 0.4% DMSO. Untreated mice, received 100μL of PBS/0.4% DMSO. Mice were feed ad-libitum and monitored closely forsign of discomfort. At the end of the survival study, tissue burdenculture was performed in mice that survived the infection. Mice weresacrificed and their organs were extracted and homogenized in 10 mlsterile PBS using homogenizer. Organ homogenates were diluted 10 timesin PBS, and 100 μL was plated on YPD agar plates and incubated at 30° C.for 72 hours for CFU count.

Toxicity

In Vitro.

The murine macrophage cell line J774.16 was maintained in DMEMcontaining 10% FBS and 1% Pen-strep. At passage #7, 10⁵ cells/well inDMEM containing 10% FBS was transferred into 96 well plates and culturedfor 14 hours for the cells to adhere to the wells. BHBM or 1 were addedto the cells at concentration ranging from 0.1 to 100 μg/ml. The wellswithout the drug served as control. The plate were incubated at 37° C.in the presence of 5% CO₂. After 12 or 24 hours, the supernatant wasremoved and 50 μL of 5 mg/ml of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)solution in PBS was added to each well and plates incubated for 4additional 4 hours. The formazan crystal formed inside the cell wasdissolved by adding 50 μL of isopropanol containing 0.1N HCL. Theoptical density was measured at 570 nm.

To determine whether BHBM or 1 toxicity was enhanced by corticosteroids,a separate set of J774.16 cells were incubated with 10 or 100 μg/ml ofDexamethasone alone or combined with either 1, 5 and 10 μg/ml of BHBM orwith 1, 5, 10 μg/ml of 1. After 24 hours, the MTT assay was performed asdescribed above.

In Vivo.

Mice toxicity studies were performed using 4-week old CBA/J female micefrom Jackson Laboratory. Five mice received 1.2 mg/kg/day of BHBM for 60days. Three control mice received a solvent injection per day. At 60day, blood was collect in two tubes: one with K₂EDTA and the otherwithout K₂EDTA to allow blood clotting. The blood clot was thencentrifuged at 1500 rpm for 10 min, serum was collected and analyzed forliver and kidney blood tests. The non-coagulated blood was used forhematocrit and blood cells analysis. These tests were done using MASCOT™HEMAVET 950FS (Drew Scientific Group, Dusseldorf, Germany)

Pharmacokinetics of BHBM

BHBM was dissolved in a mixture of cremophore:ethanol (1:1) to prepare10 mg/ml stock solution. The stock solution was diluted in PBS to obtain200 μg/ml and 400 μg/ml solutions for both IV and IP administrations inC3H/HeN mice (n=3). BHBM was administered to control healthy mice orimmunocompromised mice infected with Pneumocystis murina at doses of 0.8mg/kg and 1.6 mg/kg via IV tail vain injection or intraperitonealinjection in 100 μL final volume. The mice from each group weresacrificed and blood samples were collected at pre-dose and 0.5, 1, 2,4, 8, 12, and 24 post-administration into K₂EDTA containing tubes. Thesamples were centrifuged immediately and plasma was collected and storedat −80° C. until analysis. Plasma samples were extracted using methylenechloride. Briefly, 50 μL of the plasma sample was taken into a glassvial and 10 μL of internal standardN′-(3-bromobenzylidene)-4-hydroxybenzohydrazide was added. After mixing,1 ml of methylene chloride was added and the samples were vortex mixedfor 30 seconds followed by centrifugation for 5 minutes. Eight hundredmicroliters of supernatant was transferred to another test tube andevaporated to dryness using a centrifugal evaporator. The residue wasreconstituted in 100 μL acetonitrile: water (50:50) solution, mixed andtransferred to mass spec vials. Separation was performed under isocraticreverse phase chromatographic condition using a water's XBridge C18column (3.5 μm; 2.1×100 mm) (Waters, Milford, Mass.), a FinniganSurveyor MS pump (ThermoFisher Scientific, Waltham, Mass.), and aFinnigan Micro AS autosampler (ThermoFisher Scientific). The mobilephase consisted of water:acetonitrile with 0.1% formic acid (50:50) ranat a flow rate of 200 μL/min. Aliquots of 5 μL were analyzed usingLTQ-FT liquid chromatography/tandem mass spectrometer (LC/MS/MS) withelectrospray source in the positive ion mode (ThermoFisher Scientific,Waltham, Mass.). The retention time of BHBM was 5.7 mins. The lowerlimit of quantification (LLOQ) was 10 ng/ml. Systemic exposure of BHBMin mice was quantified by calculating the AUC of the drug from pre-doseto the end of the dosing interval (AUC_(0-t)) using the lineartrapezoidal rule by non-compartmental analysis employing PhoenixWinNonlin 6.3 (Pharsight Corp, Mountain View, Calif.). The eliminationhalf-life (t_(1/2)) was calculated as 0.693/λ_(z), where λ_(z) is theterminal elimination rate constant. Bioavailability of IP route wascalculated.

Lipid Mass Spectrometry

For lipid analysis by mass spectrometry, fungal cells (C. neoformans orC. albicans) were grown in YNB and incubated with BHBM or 1 as explainedfor the in vivo labeling (except that tritiated palmitate was notadded), for 6 hrs. Samples without drug were included as control. Beforelipid extraction, lipid internal standards (C17 ceramide and C17sphingosine) were added. Lipids were then extracted following themethods of Mandala and Bligh and Dyer and one fourth of the sample wasaliquoted for determination of the inorganic phosphate. The remainder ofthe sample was subjected to base hydrolysis and then analyzed usingLC/MS. Results were normalized with the inorganic phosphate levels.

In Vitro Activity of Gcs1

For the in vitro Gcs1 assay, C. neoformans wild-type (WT) or the Δgcs1cells were grown in YPD broth overnight at 30° C. in a shaker incubator.Cells were washed with sterile water and then lysed by bead beating inpresence of glass bead and protease cocktail inhibitor, as described(Liberto, C. et al. 2001). Next, 800 μg of cell lysate was incubatedwith 0.3 mM C16 ceramide (C16-R—OH) and in the presence or absence ofBHBM. The mixture was subjected to 3 cycles of sonication (20 sec) andvortexing (5 sec). Next, 8 μM of radiolabelled UDP-¹⁴C-Glucose (AmericanRadiolabeled Chemical) was added and, after brief vortexing, the tubeswere incubated at 37° C. for 45 min. The reaction was stopped by adding0.9 ml of 0.45% NaCl solution containing chloroform:methanol 2:1. Theorganic phase was collected in a glass tube and flushed with nitrogen.The sample was dried and resuspended in chloroform:methanol 1:1. Samplewas then loaded on a TLC plate using by chloroform:methanol:water as themobile phase.

Yeast Library Screening

Variomics Library:

The screening of the Saccharomyces cerevisiae genome-wide variomicslibraries for potential BHBM resistant clones was performed as describedpreviously (Huang, Z. et al. 2013) but with slight modifications. About6×10⁷ haploid cells was plated on solid SC-Ura medium buffered withHEPES at pH 7.0, which contained BHBM at a concentration of 20 μM (˜7μg/ml) and incubated at 30° C. for 3 days.

HIP-HOP Library:

The yeast deletion collection used here comprises of approximately 5900individually barcoded heterozygous diploid strains (HaploInsufficiencyProlifing) and ˜4800 homozygous diploid strains (HOmozygous deletionProfiling) (Pierce, S. E. et al. 2007). Pools of approximately equalstrain abundance were generated by robotically pinning (S and PRobotics, Ontario, Canada) each strain (from frozen stocks) onto YPDagar plates as arrays of 384 strains/plate. After two days of growth at30° C., colonies were collected from plates by flooding with YPD andaliquoted at optical density of 2 (at 600 nm). The fitness of eachstrain in each experimental pool was assessed as described (Pierce, S.E. et al. 2007). The dose of BHBM that resulted in 15% growth inhibitionin BY4733 (the parent strain of the yeast deletion collection) wasdetermined by performing a dose response over the course of 16 h ofgrowth at 30° C. Screens of the homozygous deletion collection wereperformed for 5 generations of growth in BHBM, and screens of theHeterozygous deletion collection were collected following 20 generationsof growth. Cells were processed as described (Proctor, M. et al. 2011).Briefly genomic DNA was extracted from each sample, subjected to PCR toamplify the unique barcode identifiers and the abundance of each barcodewas determined by quantifying the microarray signal as described. Aranked list of all genes in the genome was generated for each experimentand then compared using gene set enrichment analysis or GSEA accordingto Lee (Lee, A Y et al. 2014).

C6-NBD-Ceramide Staining

The Golgi apparatus of C. neoformans and C. albicans was stained withC6-NBD-ceramide using a previously described protocol (Kmetzsch, L. etal. 2011), based on the property that this fluorescent lipid accumulatesat the Golgi of either living or fixed cells (Pagano R. E. et al. 1989).Control or BHBM-treated (4 μg/ml) yeast cells were fixed with 4%paraformaldehyde in PBS. Cell suspensions were then washed with the samebuffer and incubated with C6-NBD-ceramide (20 mM) for 16 h at 4° C. Thecells were then incubated with bovine serum albumin (BSA, 1%) at 4° C.for 1 h to remove the excess of C6-NBD-ceramide. After washing with PBS,the cells were incubated with 10 μg/ml DAPI (Sigma-Aldrich, St. Louis,USA) for 30 min at room temperature. The cells were washed again withPBS and stained cell suspensions were mounted over glass slides asdescribed above and analyzed under an Axioplan 2 (Zeiss, Germany).

Statistical Analysis

Statistical analysis for survival studies was performed usingStudent-Newman-Keuls t test for multiple comparisons using INSTAT.Statistical analysis for tissue burden and for trophic form and ascicounts was performed using the analysis of variance (ANOVA). Additionalstatistic was performed using Student t test.

Comparison Studies

For survival studies, 4-week old CBA/J female mice (Jackson Laboratory,Bar Harbor, Me.) were used. Total of forty mice were infected by tailvein injection of 200 μL containing 10⁵ cells of C. neoformans H99 andwere randomly separated into 5 groups (8 mice per group). Treatmentstarted within 2 hours of infection. The treated mice received anintraperitoneal injection of 1.2 mg/kg/day of BHBM, 1, amphotericin B or10 mg/kg/day of fluconazole in 100 μL final volume of PBS containing0.4% DMSO. Untreated mice, received 100 μL of PBS/0.4% DMSO. Mice werefed ad-libitum and monitored closely for sign of discomfort andmeningitis. Mice showing abnormal gait, lethargy, tremor, significantloss of body weight, or inability to reach water or food were sacrificedand survival was counted until that day.

Sample Preparation for Transmission Electron Microscopy (TEM)

Sample preparation for Transmission electron Microscopy (TEM) wasperformed similar to the methods of Heung (Heung et al. 2005) with minormodifications. Briefly, C. neoformans (H99) were grown in YNB (pH=7.4)at 37° C. and 5% CO₂ and treated for 6 hours with either BHBM or 1 (4μg/mL), non-treated cells were also included as control. The cells werepelleted at 3000 rpm (1700 g) and fixed with 2% EM glutaraldehyde in PBSsolution for 1 hour. Samples were then washed in PBS, placed in 1%osmium tetroxide in 0.1M PBS, dehydrated in a graded series of ethylalcohol and embedded in Embed812 resin. Ultrathin sections of 80 nm werecut with a Leica EM UC7 ultramicrotome (Leica Microsystems Inc., BuffaloGrove, Ill.) and placed on uncoated mesh copper grids. Sections werethen counterstained with uranyl acetate and lead citrate and viewed witha FEI Tecnail2 BioTwinG2 electron microscope (FEI, Hillsboro, Oreg.)Transmission Electron Microscope (TEM). Digital images were acquiredwith an AMT XR-60 CCD Digital Camera system.

BHBM Pre-Screen

For the BHBM revertant screen, the drug-sensitive RY00622 haploid strainwas used (Suzuki, Y., et al. 2011). To determine the IC₁₀₀ dose of BHBM(at which yeast cell growth is inhibited at 100% upon drug exposure), 20ul of RY00622 cells (at OD₆₀₀ 1⁻⁴) were plated on solid syntheticcomplete (SC) media alone, with DMSO, or with a range of BHBM doses(0.2, 0.4, 0.8, 1.6 and 3.2 mM) in a 46-well plate. The plate wasincubated for 2 days at 30° C. in the dark.

BHBM Revertant Screening Assay

RY00622 cells were cultured to mid-log phase (˜OD₆₀₀ 0.5) in liquid SCmedia before adjusting the cell density to 1×10⁶ cells/ml (equivalent toOD₆₀₀˜0.1). One ml of cells was plated on solid SC media containing DMSOsolvent control (0.26% v/v) or BHBM (at 0.4 mM IC100 dose) and incubatedat 30° C. in the dark. A lawn of cells grew on the solvent control,while only a single BHMB-resistant colony was identified after 9 days.Longer incubation did not result in the appearance of further resistantclones. To confirm BHBM resistance, single colonies isolated from theBHBM containing SC media were plated onto fresh solid SC mediumcontaining 0.4 mM BHBM and incubated for 2 days at 30° C. in the dark.Robust BHBM-resistant cells were seen.

Yeast Genomic DNA Preparation

Genomic DNA was extracted from RY00622 and BHBM-resistant cells usingthe Puregene kit (Qiagen), according to the manufacturer's instructions.

Next-Generation Sequencing of BHBM-Resistant RY00622

Genomic DNA was quantified using Qubit fluorometry (Life Technologies)and diluted for sequencing library preparation using Nextera XT librarypreparation kit according to the manufacturer's instructions (Illumina).Libraries were pooled and sequenced on a single MiSeq lane, generatingpaired-end 150 bp reads.

Mapping & Variant Calling

Raw FASTQ paired-end reads for the parent (RYO0622) and the revertantwere independently aligned to NCBI sacCer3(genbank/genomes/Eukaryotes/fungi/Saccharomyces_cerevisiae/SacCer_Apr2011)reference genome using bwa mem v0.7.4-r385 with the −M flag to markshorter split hits as secondary for compatibility with Picard (Li, H. &Durbin, R. 2009). Resultant SAM files were converted to BAM format usingsamtools v1.1 and sorted by coordinate using Picard v1.96 (SortSam)(http://picard.sourceforge.net). PCR duplicate reads were filtered outusing Picard MarkDuplicates (10.24% estimated duplication) and indexedusing Picard BuildBamIndex. To call single nucleotide variants (SNVs),we ran the GATK Unified Genotyper v2.1-8 (McKenna, A., et al. 2010) withthe NCBI sacCer3 reference genome, stand_call_conf=30, andstand_emit_conf=10 (DePristo, M. A., et al. 2011). The ploidy parameterwas set to 1 since the parent and revertant are in haploid state. Sincea database of known indels and known SNPs was not available, we did notperform re-alignment around known indels and quality scorerecalibration.

TEM.

Sample preparation for Transmission electron Microscopy (TEM) wasperformed similar to the methods of Hueng et al. with minormodifications (Heung, L. J. et al 2005). Briefly, C. neoformans (H99)were grown in YNB (pH=7.4) at 37° C. and 5% CO₂ and treated for 6 hourswith either BHBM or 1 (4 μg/mL), non-treated cells were also included ascontrol. The cells were pelleted at 3000 rpm (1700 g) and fixed with 2%EM glutaraldehyde in PBS solution for 1 hour. Samples were then washedin PBS, placed in 1% osmium tetroxide in 0.1M PBS, dehydrated in agraded series of ethyl alcohol and embedded in Embed812 resin. Ultrathinsections of 80 nm were cut with a Leica EM UC7 ultramicrotome (LeicaMicrosystems Inc., Buffalo Grove, Ill.) and placed on uncoated meshcopper grids. Sections were then counterstained with uranyl acetate andlead citrate and viewed with a FEI Tecnail2 BioTwinG2 electronmicroscope (FEI, Hillsboro, Oreg.) Transmission Electron Microscope(TEM). Digital images were acquired with an AMT XR-60 CCD Digital Camerasystem.

Generation of BHBM-Resistant Strains.

For the generation of BHBM-resistant strains, the drug-sensitive S.cerevisiae RYO0622 haploid strain was used (Suzuki, Y. et al. 2011).Prescreening studies were performed to determine the IC₁₀₀ dose of BHBMfor this strain (the 100% inhibitory concentration [IC₁₀₀] at which 100%yeast cell growth is inhibited upon drug exposure). For this screening,20 μl of RYO0622 cells (at an OD₆₀₀ of 10⁻⁴) were plated on solidsynthetic complete (SC) medium alone or with DMSO or with various BHBMconcentrations (67, 133, 266, 533, and 1,066 μg/ml) in a 48-well plate.The plates were incubated for 2 days at 30° C. in the dark. Thesestudies revealed an IC₁₀₀ dose of 133 μg/ml.

Screening for the BHBM-resistant mutants was performed by growing theRY00622 cells to mid-log phase (OD₆₀₀ of ˜0.5) in liquid SC mediumbefore adjusting the cell density to 1×10⁶ cells/ml (equivalent to anOD₆₀₀ of ˜0.1). One milliliter of cells was plated on solid SC mediumcontaining DMSO solvent control (0.26% [vol/vol]) or BHBM (133 μg/mlIC₁₀₀ dose) and incubated at 30° C. in the dark. A lawn of cells grew onthe solvent control, while seven BHMB-resistant colonies were identifiedafter 9 days. Longer incubation did not result in the appearance offurther resistant colonies. To confirm BHBM resistance, single coloniesisolated from the BHBM-containing SC medium were plated onto fresh solidSC medium containing 133 μg/ml BHBM and incubated for 2 days at 30° C.in the dark. Robust BHBM-resistant cells were seen.

Next-Generation Sequencing of BHBM-Resistant Strains.

Genomic DNA was extracted from RY00622 and BHBM-resistant cells using astandard yeast DNA extraction protocol (Hoffman, C. S. et al. 1987).Genomic DNA samples were quantified using Qubit fluorometry (LifeTechnologies) and diluted for sequencing library preparation using aNextera XT library preparation kit according to the manufacturer'sinstructions (Illumina, San Diego, Calif.). For the initial round ofsequencing, individual sequencing libraries were prepared for the parentand a single BHBM-resistant clone. These libraries were pooled andsequenced on a single MiSeq lane (Illumina), generating paired-end150-bp reads. Further BHBM-resistant colonies were obtained in a secondscreen, and their DNAs were pooled at equal concentrations beforepreparation of a single sequencing library for the pool. This pool wassequenced alongside a new library for the parent strain on a singleHiSeq 2500 lane (Illumina), generating paired-end 100-bp reads.

Mapping and Variant Calling.

Raw FASTQ paired-end reads for the parent (RYO0622) and theBHBM-resistant pool were independently aligned to the NCBI sacCer3reference genome using bwa mem v0.7.4-r385 (Li, R., Yu, C. et al. 2009)with the −M flag to mark shorter split hits as secondary forcompatibility with Picard. Resultant SAM files were converted to BAMformat using samtools v1.1 and sorted by coordinate using Picard v1.96(SortSam). PCR duplicate reads were filtered out using PicardMarkDuplicates and indexed using Picard BuildBamIndex. To call singlenucleotide variants (SNVs), the GATK Unified Genotyper v2.1-8 (McKenna,A. et al. 2010) was ran with the NCBI sacCer3 reference genome,stand_call_conf=30, and stand_emit_conf=10 (DePristo, M. A. et al.2011). The ploidy parameter was set at 1, since the parent and resistantstrains are in haploid state. Realignment around known indels andquality score recalibration was not performed, since a database of knownindels and known single nucleotide polymorphisms (SNPs) is notavailable.

Validation of BHBM-Resistant Yeast Mutants.

Four yeast genes (ALP5, COS111, MKK1, and STE2) were selected based onthe high-quality variant calls present in the BHBM-resistant pool. Toconfirm BHBM resistance, the individual haploid Δap15, Δcos111, Δmkk1and Δste2 deletion mutants were assayed for growth fitness aftertreatment with BHBM. Unrelated drug controls, including methyl methanesulfonate (MMS) (cytotoxic) and fluconazole (antifungal) were assayed inparallel. Strains were cultured to mid-log phase (OD₆₀₀ of ˜0.5) inliquid YPD medium before adjusting the cell density to an OD₆₀₀ of0.0625 with YPD medium. The cells were transferred to 96-well platescontaining 100 μl of YPD with DMSO solvent control (2% [vol/vol]), BHBM(6 to 733 μg/ml), MMS (10 μg/ml to 625 μg/ml), or fluconazole (2 to 306μg/ml) and incubated at 30° C. for 24 h. The fitness of individualstrains was measured using a spectrophotometer plate reader (TecanGENios, Chapel Hill, N.C.) to read OD₆₀₀ over 24 h as a proxy for cellgrowth. Relative growth inhibition was calculated by the average rateafter normalizing the OD₆₀₀ values in drug wells against the DMSOcontrol wells on each assay plate.

Example 1. Screening the ChemBridge DIVERSET-CL Library

It was previously shown that a C. neoformans Δgcs1 mutant lacking GlcCercannot grow in neutral and alkaline pH at 37° C. and 5% CO₂. Thus,49,120 compounds were screened for those that would inhibit growth of C.neoformans under these conditions (FIG. 8). Plates were incubated at 37°C. and 5% CO₂ to mimic the environmental conditions found in the hostduring infection. For the first screening, a total of 4,912 inhibitoryassays were performed (10 compounds are mixed in each well in thislibrary). Following this screening, 129 cocktails/wells in which the ODreading was less than 80% of that obtained in the control well wereselected. The 1290 compounds were then identified using the ChemBridgedatabase and screened individually using minimum inhibitoryconcentration (MIC) studies. MIC studies were performed first atalkaline pH (7.4) and then at neutral pH (7.0) and compounds inhibitingthe growth of C. neoformans at both pH levels were selected. For allcompounds, 11 dilutions (from 32 to 0.03 μg/ml) were tested andcompounds showing a MIC₈₀≤1 μg/ml were chosen. Out of 1290, 220compounds were selected. Next, these compounds were screened at acidicpH (4.0) and 18 compounds that were inactive at this pH (MIC>32 μg/ml)were selected. These 18 compounds were then tested in an in vivolabeling assay for the inhibition of GlcCer synthesis. Two compounds (ID#5271226 and #5285729, ChemBridge Online Chemical Store, San Diego,Calif., USA) were found to significantly inhibit the synthesis of GlcCerin C. neoformans cells but not in mammalian (J774) cells (FIG. 1A).Compound #5271226 was identified asN′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) andcompound #5285729 was identified as3-bromo-N′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (1). (FIG. 1Band FIGS. 8 and 9). Both compounds were fungicidal—MFC values were 4μg/ml for BHBM and 1.2 μg/ml for 1 (FIG. 1B).

Example 2. In Vitro Antifungal Activity of BHBM

BHBM was tested against a variety of clinically relevant fungi such asC. neoformans, Cryptococcus gattii, Candida albicans, Candida krusei,Candida glabrata, Candida parapsilosis, Candida guilliermondii,Aspergillus fumigatus, Rhizopus oryzae, Blastomyces dermatitis,Histoplasma capsulatum, Coccidioides spp., Paecilomyces variotii,Pneumocystis murina and Pneumocystis jiroveci. The results areillustrated in Table 1 and in the Table 2. Fluconazole resistantclinical isolates were also included. BHBM showed good in vitro activityagainst C. neoformans, C. gattii, R. oryzae, B. dermatitis, and H.capsulatum (Table 1). The compound was moderately active against C.krusei, C. glabrata, C. guilliermondii, A. fumigatus and Coccidioidesspp. (MIC=8-16 μg/ml) and, in general, not active against C. albicans,C. parapsilosis and P. variotii. All Fluconazole-resistant C. neoformansstrains were sensitive to BHBM. BHBM showed high activity against P.murina and very marked activity against P. jiroveci (Table 1). Bothcompounds were also highly synergistic when combined with Fluconazole(FIC indices 0.38 and 0.47 for BHBM and 1 respectively), andAmphotericin B (FIC indices 0.75 and 0.77 for BHBM and 1, respectively)and additive when combined with caspofungin (FIC index 1 for both BHBMand 1) (FIG. 11). BHBM was synergistic and 1 was additive when combinedwith Tunicamycin. Neither of the drugs were active against bacteria(MIC>160 μg/ml) (FIG. 12). Next, it was tested whether resistance tothese compounds can be developed by incubating C. neoformans cells todrug concentrations below the MIC. The results illustrated in FIG. 13clearly show that, as compared to fluconazole, C. neoformans cells donot develop resistance to either BHBM or 1 after 15 days of passages.

TABLE 1 In vitro antifungal activities of N′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) determined by theminimum inhibitory concentration (MIC) against several fungal clinicalisolates and reference strains, and by the percentage inhibition of ATP(IC₅₀) against Pneumocystis murina/Pneumocystis jiroveci. MIC range MICrange Species/strain (n) (μg/mL) (μg/mL) Cryptococcus neoformans 0.25-8 Candida glabrata (3)^(#) 4->32 (13)^(&) Cryptococcus neoformans (8)^(#S)0.5-2 Candida parapsilosis (3)^(#) >32 Cryptococcus neoformans (4)^(#R)  1-2 Candida parapsilosis QC^(&) >16 Cryptococcus gattii (10)^(&) 0.5-2Candida guilliermondii (3)^(&) 2->16 Cryptococcus gattii (3)^(#) 0.5-1Aspergillus fumigatus (1)^(#)  8 Candida albicans (3)^(&) >32Aspergillus fumigatus (3)^(&) >16 Candida albicans (5)^(#) >32 Rhizopusoryzae (3)^(&)  2 Candida krusei (1)^(#)  32 Blastomyces dermatitis(10)^(&) 0.5-1    Candida krusei ATCC 6258^(#)  16 Histoplasmacapsulatum (10)^(&) 0.125-1     Candida krusei QC^(&)  8 Coccidioidesspp (10)^(&) 8-16  Candida glabrata (10)^(&) 0.125-2  Paecilomycesvariotii QC^(&) >16 Agent Day 1 Day 2 Day 3 Day 4 Pentamidine 1 μg/mL78.21/45.70 80.30/86.47 74.07/84.21 27.79/81.44 BHBM 100 μg/mL96.98/98.86 98.90/98.63 99.42/98.08 91.47/98.91 BHBM 10 μg/mL88.22/96.96 94.59/97.76 93.57/94.78 66.73/90.14 BHBM 1 μg/mL 66.16/51.4887.58/92.63 74.57/87.81 44.53/76.96 BHBM 0.1 μg/mL  6.64/16.1576.03/26.22 63.84/39.09 16.55/46.25 BHBM IC₅₀, P. murina{circumflex over( )}  1.02 μg/mL <0.01 μg/mL <0.01 μg/mL  2.02 μg/mL BHBM IC₅₀, P.jiroveci ^($) 0.912 μg/mL 0.159 μg/mL 0.074 μg/mL 0.072 μg/mL MIC,minimum inhibitory concentration; IC, inhibitory concentration;^(&)Fungus Testing Lab strains (see Supplementary Table 1); ^(#)MDP Labstrains (see Supplementary Table 1); ^(S)clinical isolates sensitive tofluconazole; ^(R)clinical isolates resistant to fluconazole; QC, qualitycontrol. {circumflex over ( )}Considered to be “highly active” (IC₅₀<0.010 μg/mL); ^($)Considered to have “very marked” activity (IC₅₀0.011-0.099 μg/mL).

Example 3. BHBM and 1 have Lethal Activity Against C. neoformans

Since both compounds were fungicidal, their killing activity wasexamined using a well-established time-kill assay. BHBM showed aconcentration-dependent killing (FIG. 2A) whereas 1 showed atime-dependent killing (FIG. 2B). Compound 1 is clearly more effectivein killing C. neoformans (100% dead cells within 24 hours) and,interestingly, the killing activity does not occur earlier than 24 hourswith higher doses (FIG. 2B). BHBM is slower than 1 in killing C.neoformans cells and requires at least 72 hours of incubation to killabout 50% of the cells (FIG. 2A). Both compounds are very stable andstill retain antifungal activity after 1 year of storage at −20° C. BHBMand 1 require 30% and 50% DMSO respectively at 1 mg/ml concentration,suggesting that their solubility is acceptable but not ideal (FIGS. 9and 10).

Next, it was tested whether BHBM could kills C. neoformans cells whenlocalized intracellularly, as this fungus is considered a facultativeintracellular pathogen and, upon phagocytosis, it is able to replicatewithin macrophages and other phagocytic cells, especially when the hostimmunity is compromised. To assess the effect of BHBM on intracellularcells, macrophages were first allowed to internalize the fungus, thenany remaining extracellular cells were washed out and BHBM was added inabsence of opsonins (e.g. complement or antibody). Eventually,intracellular fungal cells will “escape” the macrophages either throughmacrophage lysis or direct extrusion (Ma, H. et al. 2006; Alvarez, M. &Casadevall, A. 2006) but, due to the lack of opsonins in the medium,these fungal cells cannot re-enter the macrophages. BHBM was found tosignificantly decrease the intracellular replication of C. neoformanscells in a dose-dependent manner, particularly after 12 and 24 hours ofincubation (FIG. 2C). These results suggest that, in addition to killingextracellular cells, BHBM also decreases the intracellular replicationof C. neoformans.

Example 4. BHBM and 1 have Potent Antifungal Activity AgainstCryptococcosis

A mice survival study was performed to test the efficacy of BHBM and 1against cryptococcosis. Five experimental groups of mice were included:mice infected and treated with vehicle (negative control); mice infectedand treated with BHBM or with 1; mice uninfected and treated with BHBMor with 1. Treatment was 1.2 mg/Kg/day for BHBM or 1 and was initiatedthe same day mice were infected with 5×10⁵ C. neoformans cellsintranasally, and continued thereafter. As illustrated in FIG. 3A, 90%of the mice treated with BHBM and 70% of the mice treated with 1survived C. neoformans infection up to 60 days whereas 100% of untreatedmice died within 33 days. At 60 days of infection, the mice thatsurvived (9 for BHBM and 7 for 1) were sacrificed and analyzed fortissue burden (FIG. 3B) and histopathology (FIG. 3C). No C. neoformanscells were found in brains, suggesting that the BHBM and 1 treatmenteither prevented the dissemination to or/and cleared C. neoformans cellsfrom the brain. Interestingly, BHBM, and more significantly 1, were ableto decrease the fungal burden in the lung compared to control mice(infected and untreated) (FIG. 3B). These results are comparable withthose obtained upon the infection caused by the mutant lacking GlcCer(Δgcs1), in which no C. neoformans cell were recovered from the brainand a low number of C. neoformans cells were recovered from the lung.This suggests that BHBM and 1 decrease virulence of C. neoformans cellsby decreasing GlcCer synthesis in C. neoformans cells during theinfection.

Histology analysis of the lungs of the mice infected with C. neoformansand not treated showed spreading of the fungal cells throughout theorgan (FIG. 3Ca). These mice also exhibited altered architecturalorganization in their brain (FIG. 3Cb). In contrast, the lungs of themice treated with either BHBM or 1 showed a granulomatous responselimited in few areas of the lung (FIGS. 3Cc and 3Ce). Brain histology ofBHBM or 1 treated mice showed no fungal cells and normal structure(FIGS. 3Cd and 3Cf, respectively).

Example 5. In Vivo Toxicity

The uninfected mice treated daily with BHBM or 1 for 60 days were alsofollowed. They appeared normal, maintained a normal weight, and showed anormal physical activity and behavior throughout the observation period.After 60 days, they were sacrificed, and blood work was performed in 5mice and histology of lungs, brains, kidney, spleen and liver in theremaining 5 mice. Three aged matched control mice (untreated anduninfected) were included for blood work (FIGS. 14, 15 and 16) andhistology analysis. Blood work showed a slight increase of liverAspartate aminotransferase (AST) (125±75 U/L) in BHBM treated micecompared untreated/healthy mice (50±9 U/L) (FIG. 14) and this was theonly parameter that was altered. Similar results were obtained with 1.Liver AST was the only parameter that was altered by the drug and allother blood parameters for liver and kidney function were normal, aswere the number of erythrocytes, thrombocytes, and leucocytes (FIGS. 15and 16). In addition, the histological analysis of the organs examinedrevealed no difference between the BHBM treated and untreated anduninfected mice.

Example 6. BHBM and 1 have Antifungal Activity Against Pneumocystosis

Since BHBM showed in vitro activity against P. murina and P. jiroveci(Table 1), its in vivo activity was tested using a well-establishedmouse model for pneumocystosis. Four experimental groups of mice wereincluded: mice infected and treated with vehicle (negative control);mice infected and treated with trimethroprim/sulfamethaxozole (T/S); andmice infected and treated with either 0.8 or 1.6 mg/Kg/twice a day ofBHBM. A set of mice was followed for survival (FIG. 4A) and a set ofmice was used for the microscopic enumeration of asci (FIG. 4B) andnuclei (FIG. 4C) in the lungs. A significant difference was observedbetween the survival of the BHBM high dose and BHBM low dose treatmentgroups. Interestingly, the low dose mice showed improved survival overthe high dose mice. There was also an improvement in survival of the lowdose mice over the vehicle treated negative controls; however, thedifference was not significant. No significant differences were seen inthe number of asci or nuclei in lung homogenates of the vehicle treatednegative control versus either of the BHBM treatment groups. There was asignificant reduction in both asci and nuclei count following 13 days ofT/S treatment versus the negative control and BHBM groups.

A different set of mice was used to test the efficacy of 1 in thismodel. Two drug doses were tested for survival (1.25 and 12.5 mg/kg/day)and 3 drug doses for tissue burden (0.6, 1.25 and 12.5 mg/Kg/day). Micesurvival results are illustrated in FIG. 4D and lung asci and nucleicounts are shown in FIG. 4E and FIG. 4F, respectively. Treatment of P.murina infected mice with 1 did not increase survival in our mouse modelof P. murina infection compared to control steroid negative controlsafter 14 days.

However, asci and troph burdens in P. murina infected mice treated withboth 1.25 mg/kg/day and 12.5 mg/kg/day of 1 for 14 days weresignificantly reduced compared to the vehicle treated negative controlgroup (FIGS. 4D and 4F). These data suggest that 1 may have a dosedependent effect in inhibiting growth of P. murina in the lung. Althoughnot statistically significant, 1 appears to display some toxicity inthis mouse model immunosuppressed with corticosteroids.

Example 7. In Vitro Toxicity of BHBM and 1

The toxicity of both BHBM and 1 was assessed against J774.16, A549 andL2 mammalian cell lines. We found no or a slight toxicity in J774 (FIG.17). EC₅₀ cytotoxicity at 48 hours was 50 μg/ml for both compoundleading to a very good EC₅₀/MIC₈₀ selectivity index (50 for BHBMand >100 for 1). A slight to moderate toxicity was observed in A549 andL2 cell line. Interestingly, mammalian cell toxicity (using J774 cellline) was enhanced when BHBM was combined with dexamethasone (FIG. 18).The EC₅₀ for BHBM dropped to 10 μg/ml when the drug was combined to 10μg/ml for dexamethasone. Also, 1 showed an increased toxicity whencombined with dexamethasone (FIG. 18). Interestingly, this increasedtoxicity was not observed when the drugs were combined with anotherimmunosuppressive drug cyclophosphamide (FIG. 19).

Because 1 showed increased toxicity when combined with corticosteroids,the efficacy against pneumocystosis was tested in an immunosuppressedmouse model in which CD4+ cells are depleted instead of using steroids.In this model, treatment with 1 improved survival and significantlydecreased the number of asci in the lung at day 14 (FIG. 20).Importantly, no signs of toxicity were observed in this immunosuppressedanimal model.

Example 8. BHBM and 1 have Antifungal Activity Against InvasiveCandidiasis

Most Candida spp examined were resistant in vitro to both BHBM and 1.

Previous studies however showed that, in this fungus, GlcCer is requiredfor virulence through a mechanism other than facilitating growth atneutral/alkaline pH (33), which is the pH used in our library screening.Thus, we assessed whether BHBM inhibits GlcCer synthesis in C. albicansand, if so, if BHBM or 1 administration will be effective againstinvasive candidiasis. It was found that the synthesis of GlcCer in C.albicans cells is inhibited in a dose dependent manner, similar to theinhibition found in C. neoformans cells (FIG. 1A). Therefore, CBA/J micewere infected intravenously with a lethal dose of C. albicans cells andthen treated them with either BHBM or 1 intraperitoneally using the samedose regimes used to treat cryptococcosis. It was found that 75% or62.5% of mice treated with 1 or BHBM, respectively, were still aliveafter 21 days of infection, whereas the average survival of untreatedmice was 10±1.7 days (FIG. 5A). Treated mice that survived the infectionwere sacrificed and their organs were excised and examined for tissueburden. The kidneys were the only organs found to be infected by C.albicans and such infection occurred only in approximately half of themice examined (FIG. 5B). These results suggest that BHBM, and moreefficiently 1, are able to clear C. albicans infection. They alsosuggest that, indeed, GlcCer is important for the pathogenicity of C.albicans, as found in previous studies (33).

Example 9. Pharmacokinetics

PK studies upon intravenous (IV) or intraperitoneal (IP) doses of BHBMwere performed in immunocompetent control healthy mice andimmunosuppressed/infected mice (FIG. 21). In immunocompetent mice, thehalf-life of BHBM upon IV high dose was found to be 1.03 hours. In thesame group, the half-life of BHBM upon IP low and high doses were 1.70and 1.43 hrs, with a bioavailability of 93.62% and 90.59% respectively(FIG. 21). In infected mice, the half-life of BHBM upon IV high dose wasfound to be 1.24 hours. In the same group, the half-life of BHBM upon IPlow and high doses were 0.79 hours and 0.84 hours with a bioavailabilityof 93.62% and 100.33% respectively (FIG. 21). Overall, the resultsindicated that systemic exposure (AUC_(0-t) and C_(max)) upon IPadministration was higher in immunosuppressed/infected group compared tonormal group. Preliminary studies on tissue distribution showed thatBHBM is found in the brain tissue, suggesting that the molecule is ableto cross the blood-brain barrier.

Example 10. Mechanism of Action

BHBM and 1 to inhibit GlcCer production in fungi and not in mammaliancells (FIG. 1A). The synthesis of GlcCer in fungi occurs in 3 steps(FIG. 24A). First, ceramide is desaturated in position 8 of thesphingosine backbone by the sphingolipid delta 8 desaturase (Sld8)producing the Δ8-ceramide. Next, the Δ8-ceramide is methylated inposition 9 of the sphingosine backbone by the sphingolipid methyltransferase 1 (Smt1), producing the Δ8-C9-methylceramide. Finally, thisceramide is than used by Gcs1 to make Δ8-C9-methyl GlcCer. Ceramide isproduced in the ER and transported to the Golgi by vesicle sorting forthe synthesis of complex sphingolipids (Funato, K. & Riezman, H. 2001;Kajiwara, K. et al. 2014; Reggiori, F. & Conzelmann, A. 1998). Both Sld8and Smt1 enzymes are not found in mammalian cells. In fact, mammaliancells make GlcCer from “ceramide” and, thus, their GlcCer is neitherdesaturaded nor methylated (Del Poeta, M. et al. 2014). Sld8, Smt1 andGcs1 are also not found in the yeast model Saccharomyces cerevisiae and,thus, this yeast does not make any GlcCer. Thus, it was evaluated as towhether BHBM targets Gcs1, Smt1 or/and Sld8 in C. neoformans.

It was found that BHBM does not inhibit the in vitro activities of Gcs1(FIG. 24B), Smt1 or Sld8 activities. These results suggest thatinhibition of GlcCer production by BHBM must occur upstream of Sld8.This hypothesis is further supported by the fact that S. cerevisiae andC. glabrata, two fungi that do not produce GlcCer, are still partiallysensitive to BHBM, although to a lesser extent than C. neoformans andother fungi. Hence, a S. cerevisiae library was used to identify thepotential pathway(s) affected by BHBM. Although S. cerevisiae does notproduce GlcCer, other genes involved in the sphingolipid pathwayupstream Sld8 are conserved between this yeast and other fungi making itan appropriate yeast system to study this pathway.

Therefore, a Saccharomyces cerevisiae genome-wide variomic library wasscreened for genes that, when mutated, might confer resistance to BHBM.Such resistance genes could define target(s) of the compound. A previousproof-of-concept study has shown that this method could be used torapidly identify targets of small molecules (Huang, Z. et al. 2011).However, BHBM-resistant clones could not be isolated despite multipleattempts. Therefore, a S. cerevisiae HIP-HOP heterozygote mutant librarywas screened (Huang, Z. et al. 2011). The rationale for using the latterfor drug-target identification is that if a gene/enzyme is targeted by adrug then the respective heterozygote mutant will be more sensitive tothe drug compared to the wild-type “diploid” strain.

Out of 5,900, 24 heterozygote mutants were found that were >2-fold moresensitive to BHBM compared to the diploid strain (FIG. 25 and Table 3).In these 24 genes, 8 are dubious open reading frames or with nohomologies in C. neoformans. Among the remaining 16 genes, 9 genes(RET2, UBP3, SEC26, PEP7, SEC31, YML018C, SNF8, GOS1, and RET3) areinvolved in the regulation of vesicle sorting or transport between Golgiand ER (Table 3). The genes identified are conserved in C. neoformansbut none of these genes have been characterized. Among the others 7, 6genes (MTW1, REG1, SPT4, SAP4, GLC7, and ACT1) regulate chromatinorganization, cell cycle progression or cell division (Table 3). Asbriefly discussed above, vesicle transport is the main mechanism bywhich ceramide is transported between the ER and Golgi for the synthesisof GlcCer and other complex sphingolipids (55-57) and indeed, the lossof GlcCer in C. neoformans (Δgcs1) results in cell cycle arrest andblock of cell division. Therefore, these data suggest that enzymesinvolved in ceramide transport may be the target(s) of BHBM resulting inpotential inhibition of the enzymes involved in the regulation of cellcycle due to the reduction in GlcCer levels.

Example 11. Biochemical and Microscopic Studies Upon Treatment with BHBM

To support the genetic data, biochemical studies were performed toobserve changes in sphingolipid level upon BHBM treatment by TLC andmass spectrometry. As illustrated in FIG. 6, GlcCer decreased anddihydroceramide, sphingosine and sphingosine-1-phosphate increased upontreatment with BHBM (FIG. 6A). These changes were confirmed by massspectrometry (FIGS. 6B, 6C and 6D). Importantly, no significant changesof GlcCer were observed in mammalian cells at early (radioactivelabeling, FIG. 26), or at late time points (fluorescent labeling, FIG.27), or by mass spectrometry. Taken together, these results suggest thatin fungal cells BHBM and 1 most likely target specifically themetabolism or/and transport of certain ceramide species which are thenused for the synthesis of GlcCer.

Example 12. Treatment of Fungal Cells with BHBM Affects Golgi Morphologyand Cellular Sterol Concentration

The well defined relationship between lipid composition, membranearchitecture and fungal lipid secretion (Huang, Z. et al. 2011;Oliveira, D. L. et al. 2009) led to the evaluatation as to whetherexposure of fungal cells to BHBM would affect Golgi morphology, sincethis organelle is a well known regulator of secretory processes.Staining of control yeast cells with C6-NBD-ceramide revealed thetypically disperse Golgi morphology of C. neoformans (FIG. 7A) (Rizzo,J. et al. 2009; Kmetzsch, L. et al. 2011). Staining of C. albicans withC6-NBD-ceramide was limited to well-defined areas of the cytoplasm. BHBMtreatment did induce significant alterations in the Golgi morphology,particularly in C. albicans, in which changes in the Golgi morphologywere dramatic, including an apparent diffusion of Golgi-relatedstructures to peripheral regions of the cell. DAPI staining revealedthat nuclear morphology remained unaffected in all cases. BHBM treatmentresulted in marked reductions of secreted vesicles as measured by theirsterol content by both C. neoformans (FIG. 7B) and C. albicans (FIG.7C). These results further confirmed that BHBM and 1 target vesiculartransport in fungi.

Example 13. Comparison of Antifungal Activity of BHBM and 1 withExisting Antifungals (Fluconazole and Amphotericin B)

Mouse survival study was performed to test the efficacy of BHBM and 1against invasive cryptococcal infection of the central nervous system(CNS). For these studies, the mice were infected intravenously resultingin rapid development of CNS infection. A triazole (fluconazole) and apolyene (amphotericin B) were also included in these studies to test theefficacy of BHBM and 1 to commonly used drugs. BHBM and 1 were used atconcentrations of 1.2 mg/kg/day, which were used for intranasalinfection studies. Amphotericin B and fluconazole were used atconcentrations of 1.2 mg/kg/day and 10 mg/kg/day, respectively. Theseconcentrations were within the concentration range previously used forstudies of cryptococcosis in murine models (0.5 to 1.5 mg/kg/day foramphotericin B and 3 to 30 mg/kg/day for fluconazole) (Barchiesi et al.2000).

All mice in the vehicle treated (control) group, succumbed to theinfection within nine days (FIG. 29). This was in agreement with thestudy of Barchiesi using the same infection method (Barchiesi et al.2000). All drugs prolonged mice survival significantly compared tocontrol although none of the drugs completely protected the mice fromthe infection. Mice treated with BHBM and 1 succumbed 11.1±3.7 and10.5±4.2 days post-infection, which was similar to the survival patternobserved with fluconazole (10±2 days post-infection) (FIG. 29). Thecomparable efficacy of BHBM and 1 against fluconazole is quitesignificant considering that the concentration of fluconazole used fortreatment was almost eight times higher than both drugs. Amphotericin Bwas the most efficient drug in prolonging mice survival (withoutcompletely protecting the mice); however, the well-established hightoxicity of this drug has made it an unfavorable choice for treatment ofcryptocococcal infections.

Since both BHBM and 1 were effective in protecting mice fromcryptococcosis following intranasal infection of mice, it was decided topush the envelope and examine their effectiveness against an invasivemodel of CNS cryptococcal infection. Fluconazole and amphotericin B wereincluded in these studies. In addition to being mainstream drugs againstcryptococcal infections; these drugs can penetrate the cerebrospinalfluid, thus providing a good bench-mark to compare the efficacy of ourdrugs against CNS infections (Paterson, L. et al. 1978; Tucker, R. etal. 1988).

Mice survival studies showed that both BHBM and 1 were similar tofluconazole in prolonging mice survival (FIG. 29). This is ofsignificant importance as the concentration of fluconazole used forthese studies was almost eight times higher than that of BHBM and 1,suggesting that these drugs could be more effective than fluconazole.Toxicity studies showed that both BHBM and 1 are well tolerated by mice(FIGS. 14-16), the low concentration of drugs needed to prolong micesurvival further bolsters these results as lower concentration can leadto lower toxicity. The observation that BHBM and 1 both prolong micesurvival following CNS infection suggests that these drugs are also ableto penetrate the CNS. This is corroborated by our preliminary studiesshowing the presence of BHBM in brain tissue.

In the mice survival study amphotericin B was the most effective drug inprotecting the mice against cryptococcosis (FIG. 29). However, it iswell established that amphotericin B suffers from drawbacks such asadverse reactions and nephrotoxicity, thus its use as a stand-alone drugis undesirable (Gallis, H. A. et al. 1990; Sawaya, B. P. et al. 1995).Amphotericin B has been used in combination therapy with other drugs forthe treatment of cryptococcosis, since our drugs have a differentmechanism of action compared to polyenes, they could be favorablepotential candidates for combination therapy with amphotericin B(Larsen, R. A. et al. 1990; Graybill, J. R. et al. 1980).

Example 14. Electron Microscopy Analysis of C. neoformans Cells Treatedwith BHBM or 1

The effect of drug treatment on cell ultrastructure was examined usingTransmission Electron Microscopy (FIG. 30). Electron micrographs showedan accumulation of large vacuoles in the cells treated with both BHBMand 1 (FIG. 30A-C). The number of vacuoles per μm² of cell surface areawere 0.84±0.58 in BHBM and 0.93±0.54 in 1 treated cells, respectivelycompared to 0.33±0.36 in untreated cells. These vacuoles accumulatenumerous small vesicles and aberrant membrane compartments similar tothose observed in S. cerevisiae mutants with deficiency in the vesicularsecretory pathway between Golgi and ER and vacuolar integrity (Hua, Z. &Graham, T. R. 2003; Salama, N. R. et al. 1997; Bryant, N. J. & Stevens,T. H. 1998; Jarmoszewicz, K. et al. 2012; Webb, G. C. et al. 1997).Interestingly, some of these compartments appear to resemble aberrantmultivesicular bodies (FIG. 30D), which in some BHBM and 1 treated cellsoccupy the entire cells (FIG. 31).

Since the HIP-HOP library screening had identified genes involved invesicle sorting and transport as potential targets of BHBM and sincefluorescent microscopy using NBD-ceramide suggest an alteration of theGolgi apparatus in treated cells, we used TEM to investigate the effectof drug treatment on vesicle transport phenomena. TEM images showed asignificant accumulation of vacuoles in the drug treated cells (FIG.30A-C). These vacuoles will eventually fuse to form giant vacuolescontaining numerous small vesicles that eventually will replace theentire structure of the cells. This suggests that after only 6 hours oftreatment the normal cellular structure is already significantlyaltered. This is not observed in untreated cells or in mammalian cellstreated with the drugs for 72 hours.

Interestingly, multiple abnormal membrane structures containing numeroussmall vesicles were also observed in BHBM and 1 treated cells (FIG.30D), which are consistent with multivesicular bodies. The shape ofvesicles in multivesicular bodies is irregular and eventually theyconfluent in large vesicular bodies that totally replace the normal cellstructure (FIG. 31).

The aberrant multivesicular bodies loaded vesicles found in the BHBM and1 treated cells also suggest that the vesicular transport between thetrans-Golgi network, the vacuole and endosome is also affected by BHBMand 1. If these vesicles are important for the transport and recyclingof methylated ceramide then GlcCer synthesis is arrested. It is alsopossible that BHBM only target the vesicular intracellular transport andthat the defect of extracellular vesicular secretion is due to the lackGlcCer, as this sphingolipid is contained in these secretory vesicles(Nimrichter, L. & Rodrigues, M. L. 2011) and thus, it may help tostabilize their membrane for proper secretion. The lack of GlcCer in thedrug-treated cells may account for this phenotype as this sphingolipidsis contained in these secretory vesicles and thus, it may help tostabilize their membrane for proper trafficking.

These abnormal membrane structures and accumulation of vacuoles havealso been reported in a S. cerevisiae mutant deficient in NEO1 gene,which is required for COPI-dependent transport from the Golgi to the ER(Bryant, N. J. & Stevens, T. H. 1998) and in a mutant deficient inSEC31, which encodes for a subunit of COPII vesicle coat proteinimportant for the ER-Golgi transport (Salama, N. R. et al. 1997) and inthe SLA2 mutant, that regulates vesicle transport and endocytosis(Mulholland, J. et al. 1997; Wesp, A. et al. 1997). Various VPS/PEPgenes have been also implicated in the regulation of the propertrafficking of vesicles from the trans-Golgi to the vacuole in S.cerevisiae (Bryant, N. J. & Stevens, T. H. 1998; Jarmoszewicz, K. et al.2012; Webb, G. C. et al. 1997) and in Candida albicans (Palmer, G. E.2011). Intriguingly, the genes that regulate COPI, COPII, andtrans-Golgi vesicular trafficking were also identified by the HIP-HOPscreening assay as the potential target(s) of BHBM (Table 2) and, asdiscussed herein, SLA2 was identified in the sequencing of theBHBM-resistant strain.

Example 15. Treatment of Fungal Cells with BHBM or 1 AffectsIntracellular Vesicular Membrane Organization and Structure

To further validate the effect of BHBM and 1 on vesicular structure andorganization, the effect of drug treatment on cell ultrastructure wasexamined using Transmission Electron Microscopy (FIGS. 7 & 30). Electronmicrographs showed an accumulation of large vacuoles in the cellstreated with both BHBM and 1 (FIG. 30A-C). The number of vacuoles perμm² of cell surface area were 0.84±0.58 in BHBM and 0.93±0.54 in 1treated cells, respectively compared to 0.33±0.36 in untreated cells.These vacuoles accumulate numerous small vesicles and aberrant membranecompartments similar to those observed in S. cerevisiae mutants withdeficiency in the vesicular secretory pathway between Golgi, ER andplasma membrane (Hua, Z. & Graham, T. R. (2003); Salama, N. R. et al.1997); Bryant, N. J. & Stevens, T. H. 1998; Jarmoszewicz, K. et al.2012; Webb, G. C., et al. 1997; Mulholland, J. et al. 1997) identifiedby the HIP-HOP and the genome sequencing of the BHBM-resistant strain.Interestingly, some of these compartments appear to resemble aberrantmultivesicular bodies (FIG. 30D), which in some BHBM and 1 treated cellsoccupy the entire cells (FIG. 31).

In order to pinpoint the fungal molecular target of BHBM, a BHBMresistant mutant was generated using the S. cerevisiae RYO0622 strain.This strain is particularly suitable for drug target discovery becausebears precise deletions of all 16 ATP-binding cassette transporters withclades associated with multidrug resistance (Suzuki, Y., et al. 2007).Following the methodology described in the method section, one stableresistant colony to BHBM was obtained, whose genome was sequenced alongwith the parent genome and compare with the genome of NCBI sacCer3.Using GATK, 53 SNVs unique to the parent, 39 unique to the revertant,and 202 shared by the parent and revertant were identified. Of the 39SNVs unique to the variant 10 were classified as low quality callsleaving 29 revertant-specific SNVs that pass the quality threshold. Outof 29, 17 mutations were eliminated because they were inconsistentlypresent (<90%) in the revertant reads or they showed a low coveragedepth (<100 reads) in the parent data. The resulting 12 mutations areillustrated in the Supplementary File RY00622-SR.xlsx. These 12mutations are localized in the intergenic region ARS209 (7 mutations),upstream the ATG start site of the OM14 gene (1 mutation), in the codingregion of the ALP1 gene (3 mutations) and SLA2 gene (1 mutation) (Table4).

TABLE 4 Sequence comparison using the Genome Analysis Toolkit (GATK) ofS. cerevisiae RYO0622 sensitive and resistant strains to BHBM. Out of~1,000,000 cells, one colony became resistant to BHBM (see methods). Thegenome of the resistant strain, along with the genome of the parentsensitive strain, was sequenced. The two genomes were then compared toNCBI sacCer3 using GATK and Strelka. The Table illustrates mutations ingenes involved in ER-Golgi vesicular trafficking and endocytosis (SLA2and ALP1). Chromosome position, Nucleotide mutations, Gene AA mutationand location Function in S. cerevisiae Homologous in C. neoformans SLA2Chr XIV 189585, C-T, Ser512Phe Synthetic Lethal with ABP1 Glucosemetabolism. CNAG_02237 hypothetical protein. Also called END4 and MOP2.Involved in actin filament organization, PMA1 localization and function,endocytosis, exocytosis, and vesicle transport. Interacts with ACT1, VPS(see HIP-HOP) and SEC family. ALP1 Chr XIV 136877 A-G, Pro253ProArginine transporter, basic amino acid permease. CNAG_07902 hypotheticalprotein. Chr XIV 136894 T-C, ALP1 has a paralog, CAN1. Interacts withthe Ile255Ile sphingolipid metabolizing gene ISC1 and it is Chr XIV136900 T-A, regulated by RIM101. Important in endocytosis and Asn261Serdrug resistance.

Strelka v1.0-14 (Saunders, C. T., et al. 2012) was run to calldifferential (somatic) variants using the parent as the “normal” sampleand the revertant as the “tumor” sample; otherwise the provided defaultconfiguration for bwa reads was used unchanged. Strelka identified 18“somatic” SNVs between the revertant and parent. Nine mutationsidentified by GATK were also confirmed by Strelka. The differencebetween Strelka and GATK may be attributable to the differences inspecificity of the methods.

The involvement of BHBM in targeting vesicle transport is furtherconfirmed by the sequencing of the S. cerevisiae resistant colony toBHBM. The revertant showed mutations in the SLA2, ALP1 genes and in fewintergenic regions. Particularly interesting is the mutation found inthe SLA2 gene (Table 4). The SLA2 (also called END4 or MOP2) generegulates vesicle transport and endocytosis in S. cerevisiae(Mulholland, J. et al. 1997; Wesp, A. et al. 1997). The temperaturesensitive Δsla2 mutant accumulates Golgi derived secretory vesiclesstrikingly similar to the vesicles observed by our electron microscopyof BHBM treated C. neoformans cells (FIG. 30). The SLA2 gene alsoregulates cell cycle progression in S. cerevisiae and C. albicans andthe Candida gene complements S. cerevisiae Δsla2 mutant, suggesting thefunction of this gene is conserved among fungi (Asleson, C. M., et al.2001; Gale, C. A. et al. 2009) and most likely in C. neoformans. TheSLA2 gene is essential for fungal growth at 37° C. (Mulholland, J. etal. 1997; Wesp, A. et al. 1997) and this explains why BHBM resistantstrains were not found in our C. neoformans screening (performed at 37°C.), but were found in S. cerevisiae screening (performed at 30° C.).

The Sla2 protein has 3 major domains: the N-terminus, the coil and theC-terminus domains (Gottfried, I. et al. 2010). The C-terminus domaincontains the I/LWEQ modules and interacts with actin (McCann, R. O. &Craig, S. W. 2010). This domain shares high similarity with themammalian counterpart protein Talin (McCann, R. O. & Craig, S. W. 1997).On the other hand, the coil domain and the N-terminus domains are highlydivergent from Talin and these domains specifically control endocytosisand vesicle transport and maintenance in yeasts. Interestingly, the coildomain interacts with Rvs167 for controlling endocytosis and vesicletransport ¹³ and our HIP-HOP analysis showed Rvs167 to be 1.7 time moresensitive to BHBM (Table 2). The coil domain is also important for thedimerization of Sla2, which appears to be required for activity(Gourlay, C. W. et al. 2003). The point mutation in the resistant colonyis localized in the coil domain resulting in changing of the amino acid512 from serine to phenylalanine. Since mutations in the coil domainsignificantly increase the half-life of Sla2 (Mulholland, J. et al.1997; Wesp, A. et al. 1997; Ynag, S. et al. 1999), it is possible thatthe mutation at 512 is a gain of function. Thus, it was proposed thatthe target of BHBM is the coil or/and the N terminus of Sla2. It ispossible that BHBM acts to break the dimer, which is reinforced orsimply prevented by the Ser512Phe mutation. These domains are fungalspecific and are different from the counterpart regions of the humanhomolog Talin. This explains the effect of BHBM in blocking vesicletransport and GlcCer synthesis in fungi but not in mammalian cells.

It is possible that the other mutations identified in the resistantstrain are also involved (Table 4). Particularly, ALP1 regulates thetransport of basic amino acids, which might be involved in regulatingfungal tolerance to alkaline environment, in which BHBM is indeedparticularly active. Also, Alp1 interacts with inositol sphingolipidphospholipase C 1 (Isc1) enzyme, but although Isc1 is an importantenzyme in the sphingolipid pathway for the generation of long chainceramide (Farnoud, A. M. et al. 2014; Henry, J. et al. 2011; Shea, J. etal. 2006; Garcia, J. et al. 2008), it is not directly involved in thesynthesis of GlcCer. Mutations or/and point mutations of these genesidentified by HIP-HOP and by the genome sequencing can be generated fortesting whether the genes are in fact targeted by the drug.

Example 16. Synthesis and In Vitro Activity and Toxicity of NewDerivatives

Although the compound BHBM is relatively safe when used alone, their invitro toxicity increases when used in combination with corticosteroids.In addition, while active against C. neoformans and most dimorphicfungi, both BHBM is less effective against certain Candida spp. and A.fumigatus. Additionally, BHBM has a short half-life in bloodstream (˜1.4hour) and, although BHBM is very soluble, it required a small amount ofDMSO (0.4%) for complete dissolution. Additional compounds weresynthesized with improved activity, improved toxicity and/or solubilityprofiles of BHBM and/or 1 (FIG. 22). It was found that compound 1 hasimproved activity (FIG. 23). Compound 9 is 50-fold more soluble than theparent 1 (FIG. 23). Compound 10 retained a potent in vitro activityagainst C. neoformans, with improved toxicity (FIG. 23).

Example 17. Additional Analogs

An additional aspect of the invention provides derivatives of compounds1, 5, 6, 9 or 10 that also inhibit fungal shingolipid synthesis and areactive as antifungal agents. These derivatives have analogous orimproved activity to any one of compounds 1, 5, 6, 9 or 10.

Example 18. Cellular Targets

To pinpoint the cellular targets of BHBM, a second approach, generationof BHBM-resistant mutants was followed. The drug-sensitive S. cerevisiaeRYO0622 strain was used for the generation of mutants (Suzuki, Y. et al.2011). A pre-screening study exposing this strain to variousconcentrations of BHBM revealed that a drug concentration of 133 μg/mLcompletely inhibited yeast growth (IC₁₀₀ dose). Incubating 10⁶ cells ofS. cerevisiae RYO0622 strain with BHBM at IC₁₀₀ dose resulted in sevenresistant colonies, the genomes of which were sequenced and comparedwith the genome of NCBI sacCer3 using the Genome Analysis Toolkit(GATK). This analysis led to the identification of mutations in fourgenes (APL5, COS111, MKK1, and STE2) that were present in all resistantmutants.

The proteins encoded by these loci are known to be involved in vesicletrafficking, budding, and cell cycle progression (Knaus, M. et al. 2007;Elia, L. et al. 1998; Merchan, S. et al. 2011; Tongm, Z. et al. 2007),which are again in agreement with the observations of cell morphologyand known phenotypes in the absence of GlcCer (Rittershaus, P. C. et al.2006; Rodrigues, M. L. et al. 2000). These findings also closely matchthe pathway proposed by the HIP-HOP analysis. Interestingly, these fourgenes each interact with UBI4 which encodes ubiquitin and which isconjugated to proteins to target them for degradation. UBI4 is a memberof the endomembrane recycling pathways as defined by Finley et al.(Finley, D. et al. 2012).

To confirm drug resistance, individual mutants along with the parentstrain were grown in the presence of various concentrations of BHBM.Various concentrations of fluconazole and methyl methane sulphonate(MMS) were used as controls (FIG. 32A-C). All individual mutants showedincreased resistance to BHBM in the range of 11 to 92 μg/mL (FIG. 32A),while all mutants showed similar susceptibility to fluconazole and MMS.The increased resistance of these mutants to BHBM treatment confirmsthat the above-mentioned genes are indeed the targets of BHBM and theabsence of these targets impairs the killing activity of the drug. It isworth noting that deep sequencing analysis also revealed the presence ofmutations in another gene SLA2, also involved in vesicular transport(Mulholland, J. et al. 1997). Although the Δsla2 mutant was notresistant to BHBM, it remains to be determined if point mutations withinthe SLA2 open reading frame do in fact confer resistance.

Discussion

There is a major clinical need for new drugs due to a dramatic increaseof morbidity and mortality by invasive fungal infections. Using C.neoformans as a model organism, a ChemBridge synthetic library of˜50,000 compounds and looked for inhibitors of cryptococcal growth atneutral and alkaline pH and select those compounds that inhibited GlcCersynthesis. Two compounds were identified (BHBM and 1) that decreased thesynthesis of GlcCer in C. neoformans but not in mammalian cells. Thesecompounds were effective in vitro against a series of pathogenic fungi,protect mice from cryptococcal meningitis, invasive candidiasis andsignificantly decrease lung burden of P. murina, the murine model ofhuman pneumocystosis. The compounds had limited toxicity in vitro, werewell tolerated in animals and possess acceptable pharmacokineticproperties. Mechanistic studies revealed that the compounds may targetenzymes involved in the transport of vesicles between ER and Golgi,which is the main mechanism by which ceramide is transported for thesynthesis of GlcCer. It was also found that these compounds may affectthe function of enzymes involved in cell cycle progression and celldivision, further confirming the key role of GlcCer in the regulation ofthese fungal cellular processes. Finally, in vitro resistance was notdeveloped and a strong synergistic or additive effect was observed.

Without being limited by a particular theory, the compounds containedherein decrease the synthesis of fungal but not mammalian GlcCer. Thisaction seems to be specific to the transport of fungal ceramide species.The compounds are active in vitro against fungi, especially C.neoformans, P. murina, P. jiroveci, R. oryzae, and dimorphic fungi. Thecompounds appear to be effective in vivo against cryptococcosis,candidiasis and also against pneumocystosis. However, some toxicityemerged when BHBM was combined with corticosteroids. The compounds donot induce resistance in vitro and they are synergistic with existingantifungals.

C. albicans is resistant in vitro but not in vivo. Studies performed inthis fungus have suggested that GlcCer is important for virulence butthrough a mechanism other than facilitating growth at neutral/alkalinepH (33), which is the pH used to screen our ChemBridge library. Hence,inhibition of GlcCer in C. albicans by BHBM does not block fungal growthin vitro. However, because the compound still decreases GlcCersynthesis, which is required for Candida virulence, the treatment iseffective in partially protecting mice from invasive candidiasis. Thesefindings support previous studies suggesting that the effect of GlcCerin vivo during Candida infection goes beyond the regulation of fungalalkaline tolerance.

A recent paper describes the synthesis of isoniazid derivatives andtheir antifungal action against H. capsulatum (de Aguiar Cordeiro, R. etal. 2014). These compounds decrease the synthesis of ergosterol in H.capsulatum, although not as dramatic as itraconazole does (de AguiarCordeiro, R. et al. 2014). In contrast, the present compounds do notdecrease the synthesis of ergosterol (FIG. 28).

The lead compounds possess a good selectivity index (EC₅₀/MIC>50)although we hope that this index can be significantly improved by thesynthesis of new derivatives. Data showing that some derivatives that wealready produced have much better solubility, at least in vitro, suggestthe possibility that compounds with higher selectivity index can beproduced. Of interest was the change in the phenolic OH group in BMBH,alkylation of this group (Compound 2) resulted in the loss of activity.This implicated that a hydrogen promoter may be relevant to activity.Replacement of the bromo-substituent by hydrogen resulted in an inactivemolecule (Compound 3, Compound 7) implying that the phenol group aloneis not sufficient and that a bulky hydrophobic group might be required.In the absence of the bromo substituent, replacement of the phenolicgroup by dimethylamino also renders an active molecule (Compound 4,Compound 8). Surprisingly, introducing two hydroxyl groups in positions2 and 4 (Compound 5, Compound 9) resulted in 32% or 9.6% antifungalactivity when based on BMBH and 1, respectively. The 2-OH group probablyforms an intra-molecular hydrogen bond to the Schiff base nitrogen.Unexpectedly, the 4-methoxy derivatives still retained antifungalactivity; the BMBH-derived analog was slightly less active (20%) thanthe hydroxyl form, whereas the activity of the 1-derived analog improvedsignificantly to 19%. Compound 5 is 30-fold more water-soluble thanBHBM.

Both BHBM and 1 inhibit GlcCer synthesis; however, this lipid is mostlikely not the only target of these compounds. In fact, the blockage offungal growth in alkaline pH due to the loss of GlcCer (Δgcs1 mutant)can be restored if Δgcs1 cells are shifted to an acidic environment(Singh A. et al. 2012). This can occur even after the cells are left incell cycle arrest for 72 hours. This means that the lack of GlcCer has a“static” effect on cell growth. But BHBM, and more efficiently 1, killsfungal cells. One explanation for this effect is found by analyzing theHIP-HOP results, in which several genes/enzymes affected by BHBM areessential (Table 3). Additionally, treatment with BHBM or 1 acutelyleads to the accumulation of sphingosines (FIG. 6A), which is highlytoxic to fungal cells (Chung, N. et al. 2001; Chung, N. et al. 2000).The accumulation of sphingosine species is not present when Gcs1 isdeleted (Rittershaus, P. C. 2006) or in mammalian cells treated withBHBM or 1. Thus, the effect of BHBM seems to go beyond the inhibition ofGlcCer and this may account for the fungal killing effect exerted by thedrugs and not by the absence of GlcCer.

In addition, the observation that BHBM reduces the intracellularproliferation of C. neoformans within macrophages (acidic environment)also suggests that the effect of the drug goes beyond its in vitroactivity at neutral and alkaline pH. Perhaps the effect of BHBM onreducing vesicular secretion, which is not linked to the pH of themedium, is responsible for the growth inhibition of internalized fungibecause secreted vesicles are virulence bags that can protects thefungus against host cells (Rodrigues, M. L. et al. 2008).

Our toxicity and PK studies revealed that both BHBM and 1 are, ingeneral, well tolerated. However, serum half-life (T_(1/2)), C_(max) andAUC_(0-t) are low at the dose regimes used. These doses were selectedbecause they were effective in protecting against C. neoformans and C.albicans infections. It is likely that soon after the administration thedrug promptly leaves the bloodstream and concentrates in specific organsand tissues. Preliminary results on tissue distribution revealed thatBHBM is found in the brain. This suggest not only that the drug crossesthe blood-brain barrier but it may also explain the low serum half-lifeand C_(max). Once maximum tolerated dosages (MTDs) are determined, PKparameters and tissue distribution will be better assessed. However, thecompounds seem to be well tolerated and, after 60 days of treatment,mice only presented a slightly increased level of liver AST.

The study and the characterization of the genes identified by theHIP-HOP screening does reveal not only the mechanism of action of thepresent compounds but also new means by which GlcCer and other complexsphingolipids (e.g. IPC) are synthesized in fungal cells. The fungalGlcCer synthetic pathway appears to be highly specialized because BHBMdoes not block the synthesis of mammalian GlcCer (FIG. 1A and FIG. 26)or the synthesis of fungal IPC (data not shown), which also occurs inthe Golgi. In fact, in addition to GlcCer, most fungi also make anotherglycosphingolipid in the Golgi called inositol phorphorylceramides (IPCsand its derivatives). These lipids are synthesized from differentceramide species named “phytoceramides” (Del Poeta, M. et al. 2014;Nimrichter, L. et al. 2011; Rittenour, W. R. et al. 2011). Since BHBMdoes not decrease the level of IPC, it suggests that the vesiclesinvolved in transporting methylated ceramides may be different thanthose transporting phytoceramides. This type of specialization seems tobe present also in mammalian cells, in which inhibition of the transportof certain species of ceramide (very long chain) decreases GlcCer butnot sphingomyelin levels (Loizides-Mangold, U. et al. 2012). This meansthat there is a high specialization in the transport of ceramide speciesbetween the ER and the Golgi and, thus, it is possible to specificallytarget one transport without affecting the other(s). The effect of thepresent compounds on ER-Golgi vesicles is supported by their additive orsynergistic action when combined with tunicamycin, an inhibitor ofN-linked glycosylation and an ER vesicle stress inducer.

The specificity in inhibiting the transport of cellular vesicles isfurther supported by the visualization of labeled ceramide in fungalcells treated and untreated with BHBM and by the inhibition of secretedvesicles (FIG. 7). These studies indicate that BHBM not only inhibitsintracellular vesicular transport of ceramide but also the secretion ofvesicles extr

In summary, new molecules were identified that target the synthesis offungal but not mammalian GlcCer. These hydrazycins have potentantifungal activity in vitro and in vivo against a variety of clinicallyimportant fungi. They also displayed synergistic action with currentantifungals, low toxicity, favorable PK parameters, and fungal specificmechanisms of action.

REFERENCES

-   Aerts A M, et al. The antifungal activity of RsAFP2, a plant    defensin from raphanus sativus, involves the induction of reactive    oxygen species in Candida albicans. J Mol Microbiol Biotechnol.    2007; 13(4):243-7.-   Albuquerque P C, et al. Vesicular transport in Histoplasma    capsulatum: an effective mechanism for trans-cell wall transfer of    proteins and lipids in ascomycetes. Cell Microbiol. 2008;    10(8):1695-710.-   Alvarez M, and Casadevall A. Phagosome Extrusion and Host-Cell    Survival after Cryptococcus neoformans Phagocytosis by Macrophages.    Curr Biol. 2006; 16(21):2161-5.-   Aoki K, Newly discovered neutral glycosphingolipids in aureobasidin    A-resistant zygomycetes: Identification of a novel family of    Gala-series glycolipids with core Gal alpha 1-6Gal beta 1-6Gal beta    sequences. J Biol Chem. 2004; 279(31):32028-34.-   Asleson, C. M., et al. Candida albicans INT1-induced filamentation    in Saccharomyces cerevisiae depends on Sla2p. Molecular and cellular    biology 21, 1272-1284 (2001).-   Barchiesi, F., et al. Interactions between triazoles and    amphotericin B against Cryptococcus neoformans. Antimicrobial agents    and chemotherapy 44, 2435-2441 (2000).-   Benfield T, et al. Second-line salvage treatment of AIDS-associated    Pneumocystis jirovecii pneumonia: a case series and systematic    review. J Acquir Immune Defic Syndr. 2008; 48(1):63-7.-   Bligh E G, and Dyer W J. A rapid method for total lipid extraction    and purification. Can J Bioch Physiol. 1959; 37; 911-7.-   Brown G D, Denning D W, Gow N A, Levitz S M, Netea M G, and White    T C. Hidden killers: human fungal infections. Sci Transl Med. 2012;    4(165):165rv13.-   Bryant, N.J. & Stevens, T. H. Vacuole biogenesis in Saccharomyces    cerevisiae: protein transport pathways to the yeast vacuole.    Microbiology and molecular biology reviews: MMBR 62, 230-247 (1998).-   Carmona E M, and Limper A H. Update on the diagnosis and treatment    of Pneumocystis pneumonia. Ther Adv Respir Dis. 2011; 5(1):41-59.-   Chamilos G, Lewis R E, and Kontoyiannis D P. Lovastatin has    significant activity against zygomycetes and interacts    synergistically with voriconazole. Antimicrob Agents Chemother.    2006; 50(1):96-103.-   Chung N, Mao C, Heitman J, Hannun Y A, and Obeid L M.    Phytosphingosine as a specific inhibitor of growth and nutrient    import in Saccharomyces cerevisiae. J Biol Chem. 2001;    276(38):35614-21.-   Chung N, and Obeid L M. Use of yeast as a model system for studies    of sphingolipid metabolism and signaling. Methods Enzymol. 2000;    311(8):319-31.-   Colosi I A, et al. Susceptibility of 100 filamentous fungi:    comparison of two diffusion methods, Neo-Sensitabs and E-test, for    amphotericin B, caspofungin, itraconazole, voriconazole and    posaconazole. Med Mycol. 2012; 50(4):378-85.-   Cushion M T, et al. Transcriptome of Pneumocystis carinii during    fulminate infection: carbohydrate metabolism and the concept of a    compatible parasite. PLoS One. 2007; 2(5):e423.-   da Silva A F, et al. Glucosylceramides in Colletotrichum    gloeosporioides are involved in the differentiation of conidia into    mycelial cells. FEBS Lett. 2004; 561(1-3):137-43.-   Dannaoui E, et al. In vitro susceptibilities of zygomycetes to    conventional and new antifungals. J Antimicrob Chemother. 2003;    51(1):45-52.-   de Aguiar Cordeiro R, et al. Synthesis and antifungal activity in    vitro of isoniazid derivatives against histoplasma capsulatum var.    capsulatum. Antimicrob Agents Chemother. 2014; 58(5):2504-11.-   de Medeiros L N, et al. Backbone dynamics of the antifungal Psd1 pea    defensin and its correlation with membrane interaction by NMR    spectroscopy. Biochim Biophys Acta. 2010; 1798(2):105-13.-   DePristo, M. A., et al. A framework for variation discovery and    genotyping using next-generation DNA sequencing data. Nature    genetics 43, 491-498 (2011).-   Del Poeta M, Nimrichter L, Rodrigues M L, and Luberto C. Synthesis    and biological properties of fungal glucosylceramide. PLoS Pathog.    2014; 10(1):e1003832.-   Del Poeta M, et al. Synergistic antifungal activities of bafilomycin    A(1), fluconazole, and the pneumocandin MK-0991/Caspofungin acetate    (L-743,873) with calcineurin inhibitors FK506 and L-685,818 against    Cryptococcus neoformans. Antimicrob Agents Chemother. 2000;    44(3):739-46.-   Farnoud, A. M., Mor, V., Singh, A. & Del Poeta, M. Inositol    phosphosphingolipid phospholipase C1 regulates plasma membrane    ATPase (Pma1) stability in Cryptococcus neoformans. FEBS letters    (2014).-   Farowski F, et al. Intracellular concentrations of micafungin in    different cellular compartments of the peripheral blood. Int J    Antimicrob Agents. 2012; 39(3):228-31.-   Farowski F, et al. Intracellular concentrations of anidulafungin in    different compartments of the peripheral blood. Int J Antimicrob    Agents. 2013; 41(4):379-82.-   Funato K, and Riezman H. Vesicular and nonvesicular transport of    ceramide from ER to the Golgi apparatus in yeast. J Cell Biol. 2001;    155(6):949-59.-   Fungal Infection Trust, How common are fungal diseases? Fungal    Research Trust 20th Anniversary Meeting. Fungal Infection Trust;    London. Jun. 18, 2011, updated December 2012.-   Gale, C. A., et al. SLA2 mutations cause SWE1-mediated cell cycle    phenotypes in Candida albicans and Saccharomyces cerevisiae.    Microbiology 155, 3847-3859 (2009).-   Gallis, H. A., Drew, R. H. & Pickard, W. W. Amphotericin B: 30 years    of clinical experience. Review of Infectious Diseases 12, 308-329    (1990).-   Garcia, J., et al. Mathematical modeling of pathogenicity of    Cryptococcus neoformans. Molecular System Biology 4, 183-195 (2008).-   Garcia-Rodas R, et al. Capsule Growth in Cryptococcus neoformans Is    Coordinated with Cell Cycle Progression. MBio. 2014; 5(3).-   Goncalves S, Abade J, Teixeira A, and Santos N C. Lipid composition    is a determinant for human defensin HNP1 selectivity. Biopolymers.    2012; 98(4):313-21.-   Gottfried, I., Ehrlich, M. & Ashery, U. The Sla2p/HIP1/HIP1R family:    similar structure, similar function in endocytosis? Biochemical    Society transactions 38, 187-191 (2010).-   Gourlay, C. W., et al. An interaction between Sla1p and Sla2p plays    a role in regulating actin dynamics and endocytosis in budding    yeast. Journal of cell science 116, 2551-2564 (2003).-   Graybill, J. R., Williams, D. M., Van Cutsem, E. & Drutz, D. J.    Combination therapy of experimental histoplasmosis and    cryptococcosis with amphotericin Band ketoconazole. Review of    Infectious Diseases 2, 551-558 (1980).-   Guery B P, et al. Management of invasive candidiasis and candidemia    in adult non-neutropenic intensive care unit patients: Part I.    Epidemiology and diagnosis. Intensive Care Med. 2009; 35(1):55-62.-   Gullo A. Invasive fungal infections: the challenge continues. Drugs.    2009; 69 Suppl 1, 65-73.-   Henry, J., Guillotte, A., Luberto, C. & Del Poeta, M.    Characterization of inositol phospho-sphingolipid-phospholipase C 1    (Isc1) in Cryptococcus neoformans reveals unique biochemical    features. FEBS letters 585, 635-640 (2011).-   Heung L J, Luberto C, and Del Poeta M. Role of sphingolipids in    microbial pathogenesis. Infect Immun. 2006; 74(1):28-39.

Heung, L. J., Kaiser, A. E., Luberto, C. & Del Poeta, M. The role andmechanism of diacylglycerol-protein kinase C1 signaling in melanogenesisby Cryptococcus neoformans. J. Biol. Chem. 280, 28547-28555 (2005).

-   Hoffman. C. S., Winston, F. A ten-minute DNA preparation from yeast    efficiently releases autonomous plasmids for transformation of    Escherichia coli. Gene 57:267-272 (1987)-   Hua, Z. & Graham, T. R. Requirement for neo1p in retrograde    transport from the Golgi complex to the endoplasmic reticulum.    Molecular biology of the cell 14, 4971-4983 (2003).-   Huang L, Morris A, Limper A H, Beck J M, and Participants ATSPW. An    Official ATS Workshop Summary: Recent advances and future directions    in pneumocystis pneumonia (PCP). Proc Am Thorac Soc. 2006;    3(8):655-64.-   Huang Z, et al. A functional variomics tool for discovering    drug-resistance genes and drug targets. Cell Rep. 2013; 3(2):577-85.-   Huang Z, et al. Sampangine inhibits heme biosynthesis in both yeast    and human. Eukaryot Cell. 2011; 10(11):1536-44.-   Ishibashi Y, et al. Quality control of fungus-specific    glucosylceramide in Cryptococcus neoformans by    endoglycoceramidase-related protein 1 (EGCrP1). J Biol Chem. 2012;    287(1):368-81.-   Jarmoszewicz, K., Lukasiak, K., Riezman, H. & Kaminska, J. Rsp5    ubiquitin ligase is required for protein trafficking in    Saccharomyces cerevisiae COPI mutants. PloS one 7, e39582 (2012).-   Kajiwara K, et al. Osh proteins regulate COPII-mediated vesicular    transport of ceramide from the endoplasmic reticulum in budding    yeast. J Cell Sci. 2014; 127(Pt 2):376-87.-   Kazanjian P, et al. Pneumocystis carinii cytochrome b mutations are    associated with atovaquone exposure in patients with AIDS. J Infect    Dis. 2001; 183(5):819-22.-   Kechichian T B, et al. Depletion of alveolar macrophages decreases    the dissemination of a glucosylceramide-deficient mutant of    Cryptococcus neoformans in immunodeficient mice. Infect Immun. 2007;    75(10):4792-8.-   Kelley C F, et al. Trends in hospitalizations for AIDS-associated    Pneumocystis jirovecii Pneumonia in the United States (1986 to    2005). Chest. 2009; 136(1):190-197.-   Kmetzsch L, et al. Role for Golgi reassembly and stacking protein    (GRASP) in polysaccharide secretion and fungal virulence. Mol    Microbiol. 2011; 81(1):206-18.-   Larsen, R. A., Leal, M. A. E. & Chan, L. S. Fluconazole Compared    with Amphotericin B plus Flucytosine for Cryptococcal Meningitis in    AIDSA Randomized Trial. Annals of Internal Medicine 113, 183-187    (1990).-   Lee A Y, et al. Mapping the cellular response to small molecules    using chemogenomic fitness signatures. Science. 2014;    344(6180):208-11.-   Levery S B, et al. Disruption of the glucosylceramide biosynthetic    pathway in Aspergillus nidulans and Aspergillus fumigatus by    inhibitors of UDP-Glc:ceramide glucosyltransferase strongly affects    spore germination, cell cycle, and hyphal growth. FEBS Lett. 2002;    525(1-3):59-64.-   Li, R, et al. SOAP2: an improved ultrafast tool for short read    alignment. BioInformatics 25:1966-1967 (2009).-   Li, H. & Durbin, R. Fast and accurate short read alignment with    Burrows-Wheeler transform. Bioinformatics 25, 1754-1760 (2009).-   Liu O W, Chun C D, Chow E D, Chen C, Madhani H D, and Noble S M.    Systematic genetic analysis of virulence in the human fungal    pathogen Cryptococcus neoformans. Cell. 2008; 135(1):174-88.-   Lobo D S, et al. Antifungal Pisum sativum defensin 1 interacts with    Neurospora crassa cyclin F related to the cell cycle. Biochemistry.    2007; 46(4):987-96.-   Loizides-Mangold U, David F P, Nesatyy V J, Kinoshita T, and    Riezman H. Glycosylphosphatidylinositol anchors regulate    glycosphingolipid levels. J Lipid Res. 2012; 53(8):1522-34.-   Luberto C, Toffaletti D L, Wills E A, Tucker S C, Casadevall A,    Perfect J R, Hannun Y A, and Del Poeta M. Roles for    inositol-phosphoryl ceramide synthase 1 (IPC1) in pathogenesis of C.    neoformans. Genes Dev. 2001; 15(2):201-12.-   Ma H, Croudace J E, Lammas D A, and May R C. Expulsion of live    pathogenic yeast by macrophages. Curr Biol. 2006; 16(21):2156-60.-   Ma L, Borio L, Masur H, and Kovacs J A. Pneumocystis carinii    dihydropteroate synthase but not dihydrofolate reductase gene    mutations correlate with prior trimethoprim-sulfamethoxazole or    dapsone use. J Infect Dis. 1999; 180(6):1969-78.-   Mandala S M, et al. The discovery of australifungin, a novel    inhibitor of sphinganine N-acyltransferase from Sporormiella    australis. Producing organism, fermentation, isolation, and    biological activity. J Antibiot (Tokyo). 1997; 50(4):339-43.-   Maschmeyer G, Haas A, and Cornely O A. Invasive aspergillosis:    epidemiology, diagnosis and management in immunocompromised    patients. Drugs. 2007; 67(11):1567-601.-   Mayr A, and Lass-Florl C. Epidemiology and antifungal resistance in    invasive Aspergillosis according to primary disease: review of the    literature. Eur J Med Res. 2011; 16(4):153-7.-   McCann, R. O. & Craig, S. W. The I/LWEQ module: a conserved sequence    that signifies F-actin binding in functionally diverse proteins from    yeast to mammals. Proceedings of the National Academy of Sciences of    the United States of America 94, 5679-5684 (1997).-   McKenna, A., et al. The Genome Analysis Toolkit: a MapReduce    framework for analyzing next-generation DNA sequencing data. Genome    research 20, 1297-1303 (2010).-   Mello Ede, O., et al. Functional expression and activity of the    recombinant antifungal defensin PvD1r from Phaseolus vulgaris L.    (common bean) seeds. BMC Biochem. 2014; 15(1):7.-   Mulholland, J., Wesp, A., Riezman, H. & Botstein, D. Yeast actin    cytoskeleton mutants accumulate a new class of Golgi-derived    secretary vesicle. Molecular biology of the cell 8, 1481-1499    (1997).-   Munoz P, Guinea J, Narbona M T, and Bouza E. Treatment of invasive    fungal infections in immunocompromised and transplant patients:    AmBiLoad trial and other new data. Int J Antimicrob Agents. 2008; 32    Suppl 2: S125-31.-   Nimrichter L, and Rodrigues M L. Fungal glucosylceramides: from    structural components to biologically active targets of new    antimicrobials. Front Microbiol. 2011; 2; 212.-   Noble S M, French S, Kohn L A, Chen V, and Johnson A D. Systematic    screens of a Candida albicans homozygous deletion library decouple    morphogenetic switching and pathogenicity. Nat Genet. 2010;    42(7):590-8.-   Nosanchuk J D, Nimrichter L, Casadevall A, and Rodrigues M L. A role    for vesicular transport of macromolecules across cell walls in    fungal pathogenesis. Commun Integr Biol. 2008; 1(1):37-9.-   Odabasi Z, Paetznick V, Rex J H, and Ostrosky-Zeichner L. Effects of    serum on in vitro susceptibility testing of echinocandins.    Antimicrob Agents Chemother. 2007; 51(11):4214-6.-   Oguro Y, Yamazaki H, Takagi M, and Takaku H. Antifungal activity of    plant defensin AFP1 in Brassica juncea involves the recognition of    the methyl residue in glucosylceramide of target pathogen Candida    albicans. Curr Genet. 2014; 60(2):89-97.-   Oliveira D L, Freire-de-Lima C G, Nosanchuk J D, Casadevall A,    Rodrigues M L, and Nimrichter L. Extracellular vesicles from    Cryptococcus neoformans modulate macrophage functions. Infect Immun.    2010; 78(4):1601-9.-   Oliveira D L, Nimrichter L, Miranda K, Frases S, Faull K F,    Casadevall A, and Rodrigues M L. Cryptococcus neoformans    cryoultramicrotomy and vesicle fractionation reveals an intimate    association between membrane lipids and glucuronoxylomannan. Fungal    Genet Biol. 2009; 46(12):956-63.-   Oura T, and Kajiwara S. Candida albicans sphingolipid    C9-methyltransferase is involved in hyphal elongation. Microbiology.    2010; 156(Pt 4):1234-43.-   Oura T, and Kajiwara S. Disruption of the sphingolipid    Delta8-desaturase gene causes a delay in morphological changes in    Candida albicans. Microbiology. 2008; 154(Pt 12):3795-803.-   Palmer, G. E. Vacuolar trafficking and Candida albicans    pathogenesis. Communicative & integrative biology 4, 240-242 (2011).-   Pagano R E, Sepanski M A, and Martin O C. Molecular trapping of a    fluorescent ceramide analogue at the Golgi apparatus of fixed cells:    interaction with endogenous lipids provides a trans-Golgi marker for    both light and electron microscopy. J Cell Biol. 1989;    109(5):2067-79.-   Perlroth J, Choi B, and Spellberg B. Nosocomial fungal infections:    epidemiology, diagnosis, and treatment. Med Mycol. 2007;    45(4):321-46.-   Peterson, L., Kelty, R., Hall, W. & Votava, H. Therapy of Candida    peritonitis: penetration of amphotericin B into peritoneal fluid.    Postgraduate medical journal 54, 340-342 (1978).-   Pierce S E, et al. Genome-wide analysis of barcoded Saccharomyces    cerevisiae gene-deletion mutants in pooled cultures. Nat Protoc.    2007; 2(11):2958-74.-   Proctor M, et al. The automated cell: compound and environment    screening system (ACCESS) for chemogenomic screening. Methods Mol    Biol. 2011; 759(239-69.-   Ramamoorthy V, et al. Sphingolipid C-9 methyltransferases are    important for growth and virulence but not for sensitivity to    antifungal plant defensins in Fusarium graminearum. Eukaryot Cell.    2009; 8(2):217-29.-   Reggiori F, and Conzelmann A. Biosynthesis of inositol    phosphoceramides and remodeling of glycosylphosphatidylinositol    anchors in Saccharomyces cerevisiae are mediated by different    enzymes. J Biol Chem. 1998; 273(46):30550-9.-   Rhome R, et al. Biosynthesis and immunogenicity of glucosylceramide    in Cryptococcus neoformans and other human pathogens. Eukaryot Cell.    2007; 6(10):1715-26.-   Rhome R, et al. Surface localization of glucosylceramide during    Cryptococcus neoformans infection allows targeting as a potential    antifungal. PLoS One. 2011; 6(1):e15572.-   Rittenour W R, et al. Control of glucosylceramide production and    morphogenesis by the Bar1 ceramide synthase in Fusarium graminearum.    PLoS One. 2011; 6(4):e19385.-   Rittershaus P C, et al. Glucosylceramide is an essential regulator    of pathogenicity of Cryptococcus neoformans. J Clin Invest. 2006;    116(6):1651-9.-   Rizzo J, et al. Role of the Aptl protein in polysaccharide secretion    by Cryptococcus neoformans. Eukaryot Cell. 2014; 13(6):715-26.-   Rodrigues M L, Nimrichter L, Oliveira D L, Nosanchuk J D, and    Casadevall A. Vesicular Trans-Cell Wall Transport in Fungi: A    Mechanism for the Delivery of Virulence-Associated Macromolecules?    Lipid Insights. 2008; 2; 27-40.-   Rodrigues M L, and Djordjevic J T. Unravelling secretion in    Cryptococcus neoformans: more than one way to skin a cat.    Mycopathologia. 2012; 173(5-6):407-18.-   Rodrigues M L, Nakayasu E S, Oliveira D L, Nimrichter L, Nosanchuk J    D, Almeida I C, and Casadevall A. Extracellular vesicles produced by    Cryptococcus neoformans contain protein components associated with    virulence. Eukaryot Cell. 2008; 7(1):58-67.-   Rodrigues, M. L., et al. Vesicular polysaccharide export in    Cryptococcus neoformans is a eukaryotic solution to the problem of    fungal trans-cell wall transport. Eukaryotic cell 6, 48-59 (2007).-   Rogers T R. Treatment of zygomycosis: current and new options. J    Antimicrob Chemother. 2008; 61 Suppl 1(i35-40.-   Rueping M J, eInvasive candidiasis and candidemia: from current    opinions to future perspectives. Expert Opin Investig Drugs. 2009;    18(6):735-48.-   Ruping M J, Vehreschild J J, and Cornely O A. Patients at high risk    of invasive fungal infections: when and how to treat. Drugs. 2008;    68(14):1941-62.-   Saito K, Takakuwa N, Ohnishi M, and Oda Y. Presence of    glucosylceramide in yeast and its relation to alkali tolerance of    yeast. Appl Microbiol Biotechnol. 2006; 69:1-7.-   Salama, N. R., Chuang, J. S. & Schekman, R. W. Sec31 encodes an    essential component of the COPII coat required for transport vesicle    budding from the endoplasmic reticulum. Molecular biology of the    cell 8, 205-217 (1997).-   Saribas Z, Yurdakul P, Cetin-Hazirolan G, and Arikan-Akdagli S.    Influence of serum on in vitro susceptibility testing of    echinocandins for Candida parapsilosis and Candida guilliermondii.    Mycoses. 2012; 55(2):156-60.-   Saunders, C. T., et al. Strelka: accurate somatic small-variant    calling from sequenced tumor-normal sample pairs. Bioinformatics 28,    1811-1817 (2012).-   Sawaya, B. P., Briggs, J. P. & Schnermann, J. Amphotericin B    nephrotoxicity: the adverse consequences of altered membrane    properties. Journal of the American Society of Nephrology 6, 154-164    (1995).-   Shea, J., Kechichian, T. B., Luberto, C. & Del Poeta, M. The    cryptococcal enzyme inositol phosphosphingolipid-phospholipase C    (Isc1) confers resistance to the antifungal effects of macrophages    and promotes fungal dissemination to the central nervous system.    Infection and immunity 74, 5977-5988 (2006).

Singh A, and Del Poeta M. Lipid signalling in pathogenic fungi. Cellularmicrobiology. 2011; 13(2):177-85.

-   Singh A, Na C, Silva L C, Prieto M, Futerman A H, Luberto C, and Del    Poeta M. Membrane lipid topography controlled by sphingolipids    regulates pathogenicity of Cryptococcus neoformans. Cellular    Microbiology. 2012; 14(4):500-16.-   Singh J, Rimek D, and Kappe R. In vitro susceptibility of 15 strains    of zygomycetes to nine antifungal agents as determined by the NCCLS    M38-A microdilution method. Mycoses. 2005; 48(4):246-50.-   Sorrell T C, Chen S C-A, Phillips P, and Marr K A. In: Heitman J,    Kozel T R, Kwon-Chung K J, Perfect J, and Casadevall A eds.    Cryptococcus: from human pathogen to model yeast. Washington, D.C.:    ASM; 2011:595-606.-   Suzuki, Y., et al. Knocking out multigene redundancies via cycles of    sexual assortment and fluorescence selection. Nature methods 8,    159-164 (2011).-   Tavares P M, et al. In vitro activity of the antifungal plant    defensin RsAFP2 against Candida isolates and its in vivo efficacy in    prophylactic murine models of candidiasis. Antimicrob Agents    Chemother. 2008; 52(12):4522-5.-   Thevissen K, et al. The plant defensin RsAFP2 induces cell wall    stress, septin mislocalization and accumulation of ceramides in    Candida albicans. Mol Microbiol. 2012; 84(1):166-80.-   Thevissen K, et al. Defensins from insects and plants interact with    fungal glucosylceramides. J Biol Chem. 2004; 279(6):3900-5.-   Toledo M S, et al. Characterization of cerebrosides from the    thermally dimorphic mycopathogen Histoplasma capsulatum: expression    of 2-hydroxy fatty N-acyl (E)-Delta(3)-unsaturation correlates with    the yeast-mycelium phase transition. Glycobiology. 2001;    11(2):113-24.-   Tucker, R., et al. Pharmacokinetics of fluconazole in cerebrospinal    fluid and serum in human coccidioidal meningitis. Antimicrobial    agents and chemotherapy 32, 369-373 (1988).-   Tuite N L, and Lacey K. Overview of invasive fungal infections.    Methods Mol Biol. 2013; 968, 1-23.-   Vallejo M C, et al. The pathogenic fungus Paracoccidioides    brasiliensis exports extracellular vesicles containing highly    immunogenic alpha-Galactosyl epitopes. Eukaryot Cell. 2011;    10(3):343-51.-   Warnecke D, and Heinz E. Recently discovered functions of    glucosylceramides in plants and fungi. Cell Mol Life Sci. 2003;    60(5):919-41.-   Webb, G. C., et al. Pep7p provides a novel protein that functions in    vesicle-mediated transport between the yeast Golgi and endosome.    Molecular biology of the cell 8, 871-895 (1997).-   Wesp, A., et al. End4p/Sla2p interacts with actin-associated    proteins for endocytosis in Saccharomyces cerevisiae. Molecular    biology of the cell 8, 2291-2306 (1997).-   World Health Organization. World Malaria Report    2013—http://www.who.int/malaria/publications/world_malaria_report_2013/en/.    Accessed Dec. 11, 2013.-   World Health Organization. Global Tuberculosis Report    2013—http://www.who.int/tb/publications/global_report/en/. Accessed    November 2013.-   Yanni S B, et al. Higher clearance of micafungin in neonates    compared with adults: role of age-dependent micafungin serum    binding. Biopharm Drug Dispos. 2011; 32(4):222-32.-   Yang, S., Cope, M. J. & Drubin, D. G. Sla2p is associated with the    yeast cortical actin cytoskeleton via redundant localization    signals. Molecular biology of the cell 10, 2265-2283 (1999).

1. A compound having the structure:

wherein R₁ is —H; R₂ is —H; R₃, R₄, R₅ and R₆ are each —H and R₇ is—CH₃, or R₃, R₄, R₅ and R₇ are each —H and R₆ is —Br; R₉, R₁₀, and R₁₁are each independently —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl,alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃, —SH,—SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or—CONR₁₄R₁₅; and R₈ and R₁₂ are each independently —H, halogen, —CN,—CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OAc, —OH,—OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, NR₁₄R₁₅,—NHCOR₁₂, or —CONR₁₄R₁₅, wherein each occurrence of R₁₃ is independentlyalkyl, alkenyl, alkynyl, aryl, or heteroaryl, wherein each occurrence ofR₁₄ is independently —H, alkyl, alkenyl, alkynyl, aryl, or heteroaryl,wherein each occurrence of R₁₅ is independently —H, alkyl, alkenyl,alkynyl, aryl, or heteroaryl, wherein when R₁, R₂, R₃, R₄, R₅, R₆, R₈,R₁₁ and R₁₂ are each —H, R₉ is —Br, and R₁₀ is —OH, then R₇ is otherthan —CH₃, or a pharmaceutically acceptable salt or ester thereof. 2.The compound of claim 1, wherein at least one of R₈, R₉, R₁₀, R₁₁, andR₁₂ are other than —H.
 3. The compound of claim 1, wherein at least twoof R₈, R₉, R₁₀, R₁₁, and R₁₂ are other than —H. 4-5. (canceled)
 6. Thecompound claim 1, wherein R₈, R₉, R₁₀, R₁₁, and R₁₂ are eachindependently —H, —Br, alkyl, —OH, —OR₁₃, or —NR₁₄R₁₅, wherein eachoccurrence of R₁₃, R₁₄, and R₁₅ is alkyl, or a pharmaceuticallyacceptable salt thereof.
 7. The compound of claim 6, wherein R₈, R₉,R₁₀, R₁₁, and R₁₂ are each independently —H, —Br, —OH, —OR₁₃, or—NR₁₄R₁₅, wherein each occurrence of R₁₃, R₁₄, and R₁₅ is alkyl, or apharmaceutically acceptable salt thereof.
 8. (canceled)
 9. The compoundof claim 1 having the structure:

wherein R₉, R₁₀, and R₁₁ are each independently —H, halogen, —CN, —CF₃,—OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,—OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, NR₁₄R₁₅,—NHCOR₁₂, or —CONR₁₄R₁₅; and R₈ and R₁₂ are each independently —H,halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl,heteroaryl, —OAc, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,—NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅, wherein each occurrence of R₁₃is independently alkyl, alkenyl, alkynyl, aryl, or heteroaryl, whereineach occurrence of R₁₄ is independently —H, alkyl, alkenyl, alkynyl,aryl, or heteroaryl, wherein each occurrence of R₁₅ is independently —H,alkyl, alkenyl, alkynyl, aryl, or heteroaryl, or a pharmaceuticallyacceptable salt thereof.
 10. The compound of claim 9, wherein R₈ and R₁₂are each independently —H, —Br, or —NR₁₄R₁₅, R₉, R₁₀, and R₁₁ are eachindependently —H, —Br, —OH, —OR₁₃, or —NR₁₄R₁₅, wherein each occurrenceof R₁₃, R₁₄, and R₁₅ is alkyl, or a pharmaceutically acceptable saltthereof.
 11. The compound of claim 1, wherein R₈ and R₁₂ are eachindependently —H, halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl,alkynyl, aryl, heteroaryl, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃,—SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅, or apharmaceutically acceptable salt thereof. 12-14. (canceled)
 15. Thecompound of claim 1 having the structure:

or a pharmaceutically acceptable salt thereof.
 16. A pharmaceuticalcomposition comprising the compound of claim 1 and a pharmaceuticallyacceptable carrier.
 17. A method of inhibiting the growth of a fungus orof inhibiting fungal shingolipid synthesis in a fungus or comprisingcontacting the fungus with an effective amount of a compound having thestructure:

wherein R₁ is H, alkyl, alkenyl, or alkynyl; R₂ is H, alkyl, alkenyl, oralkynyl; R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,—CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,—OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or—CONR₁₄R₁₅; R₈ and R₉ are each independently —H, halogen, —CN, —CF₃,—OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,—OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, NR₁₄R₁₅,—NHCOR₁₂, or —CONR₁₄R₁₅; R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂,alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃,—SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NHCOR₁₂, or —CONR₁₄R₁₅;and R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃, —OCF₃,—NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃,—COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, NR₁₄R₁₅,—NHCOR₁₂, or —CONR₁₄R₁₅, wherein each occurrence of R₁₃ is independentlyalkyl, alkenyl, alkynyl, aryl, or heteroaryl, wherein each occurrence ofR₁₄ is independently —H, alkyl, alkenyl, alkynyl, aryl, or heteroaryl,wherein each occurrence of R₁₅ is independently —H, alkyl, alkenyl,alkynyl, aryl, or heteroaryl, or a pharmaceutically acceptable salt orester thereof, so as to thereby inhibit the growth of the fungus orinhibit fungal shingolipid synthesis in the fungus, wherein when thefungus is Cryptococcus neoformans, then when R₁, R₂, R₃, R₄, R₅, R₆, R₈,R₁₁, R₁₂ are each —H, R₉ is —Br, and —R₁₀ is —OH or —OCH₃, then R₇ isother than —CH₃; and when R₁, R₂, R₃, R₄, R₅, R₈, R₉, R₁₁, and R₁₂ areeach —H, and —R₁₀ is OH, then R₆ and R₇ are other than —H and —CH₃, or—Br, and H, respectively.
 18. (canceled)
 19. A method of inhibitingfungal shingolipid synthesis in a fungus in a mammal withoutsubstantially inhibiting mammalian shingolipid synthesis or ofinhibiting the growth of or killing a fungus in a subject afflicted witha fungal infection comprising administering to the mammal or subject aneffective amount of a compound having the structure:

wherein R₁ is H, alkyl, alkenyl, or alkynyl; R₂ is H, alkyl, alkenyl, oralkynyl; R₃, R₄, R₅, R₆, and R₇ are each independently —H, halogen, CN,—CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,—OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —NH₂, —NHR₁₃, —NR₁₄R₁₅, —NHCOR₁₂, or—CONR₁₄R₁₅; R₈ and R₉ are each independently —H, halogen, —CN, —CF₃,—OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc,—OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, NR₁₄R₁₅,—NHCOR₁₂, or —CONR₁₄R₁₅; R₁₀ is —H, halogen, —CN, —CF₃, —OCF₃, —NO₂,alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃, —COR₁₃,—SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, —NHCOR₁₂, or —CONR₁₄R₁₅;and R₁₁ and R₁₂ are each independently —H, halogen, —CN, —CF₃, —OCF₃,—NO₂, alkyl, alkenyl, alkynyl, aryl, heteroaryl, —OH, —OAc, —OR₁₃,—COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂, —NHR₁₃, NR₁₄R₁₅,—NHCOR₁₂, or —CONR₁₄R₁₅, wherein each occurrence of R₁₃ is independentlyalkyl, alkenyl, alkynyl, aryl, or heteroaryl, wherein each occurrence ofR₁₄ is independently —H, alkyl, alkenyl, alkynyl, aryl, or heteroaryl,wherein each occurrence of R₁₅ is independently —H, alkyl, alkenyl,alkynyl, aryl, or heteroaryl, or a pharmaceutically acceptable salt orester thereof, so as to thereby inhibit fungal shingolipid synthesis inthe fungus in the mammal without substantially inhibiting mammalianshingolipid synthesis or inhibit the growth of or kill the fungus in thesubject afflicted with the fungal infection, wherein when the fungus isCryptococcus neoformans, then when R₁, R₂, R₃, R₄, R₅, R₆, R₈, R₁₁, R₁₂are each —H, R₉ is —Br, and —R₁₀ is —OH or —OCH₃, then R₇ is other than—CH₃; and when R₁, R₂, R₃, R₄, R₅, R₈, R₉, R₁₁, and R₁₂ are each —H, and—R₁₀ is OH, then R₆ and R₇ are other than —H and —CH₃ or —Br and H,respectively.
 20. (canceled)
 21. The method of claim 17, wherein thecompound has the structure:

or a pharmaceutically acceptable salt thereof.
 22. The method of claim17, further comprising contacting the fungus with an amount of ananti-fungal agent.
 23. The method of claim 22, wherein the anti-fungalagent is fluconazole, amphotericin B, caspofungin, tunicamycin oraureobasidin A.
 24. The method of claim 17, wherein the fungus isCryptococcus gattii, Candida albicans, Candida krusei, Candida glabrata,Candida parapsilosis, Candida guilliermondii, Aspergillus fumigatus,Rhizopus oryzae, Rhizopus spp., Blastomyces dermatitis, Histoplasmacapsulatum, Coccidioides spp., Paecilomyces variotii, Pneumocystismurina, Pneumocystis jiroveci, Histoplasma capsulatum, Aspergillus spp.,a dimorphic fungi or a mucorales fungi.
 25. The method of claim 17,wherein the fungal shingolipid is glucosylceramide (GlcCer). 26-29.(canceled)
 30. The method of claim 17, wherein the compound has thestructure:

or a pharmaceutically acceptable salt thereof. 31-36. (canceled)
 37. Themethod of claim 17, wherein the fungus is Cryptococcus Neoformans. 38.The method of claim 17, wherein R₈ and R₁₂ are each independently —H,halogen, —CN, —CF₃, —OCF₃, —NO₂, alkyl, alkenyl, alkynyl, aryl,heteroaryl, —OAc, —OR₁₃, —COR₁₃, —SH, —SR₁₃, —SO₂R₁₃, —SO₂NR₁₄R₁₅, —NH₂,—NHR₁₃, NR₁₄R₁₅, —NHCOR₁₂, or —CONR₁₄R₁₅, or a pharmaceuticallyacceptable salt thereof.